Amino acid sequences directed against a metalloproteinase from the ADAM family and polypeptides comprising the same for the treatment of ADAM-related diseases and disorders

ABSTRACT

The present application relates to amino acid sequences that are directed against and/or that can specifically bind to metalloproteinases from the ADAM family, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences, and to methods of preparing the same.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. §371 of international application PCT/EP2007/064244, filed Dec. 19, 2007, which was published under PCT Article 21(2) in English, and claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application Ser. No. 60/875,834, filed Dec. 19, 2006, the disclosures of which are incorporated by reference herein in their entireties.

The present invention relates to amino acid sequences that are directed against (as defined herein) metalloproteinases from the ADAM family, as well as to compounds or constructs, and in particular proteins and polypeptides, that comprise or essentially consist of one or more such amino acid sequences (also referred to herein as “amino acid sequences of the invention”, “compounds of the invention”, and “polypeptides of the invention”, respectively).

The invention also relates to nucleic acids encoding such amino acid sequences and polypeptides (also referred to herein as “nucleic acids of the invention” or “nucleotide sequences of the invention”); to methods for preparing such amino acid sequences and polypeptides; to host cells expressing or capable of expressing such amino acid sequences or polypeptides; to compositions, and in particular to pharmaceutical compositions, that comprise such amino acid sequences, polypeptides, nucleic acids and/or host cells; and to uses of such amino acid sequences or polypeptides, nucleic acids, host cells and/or compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes mentioned herein.

Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein.

The ADAM metalloproteinases disintegrins form a well-known family of proteases (or proteinases) that mediates ectodomain shedding. Many of the processing events that include the release of cytokines, shedding of cell surface molecules, release of growth factors and cleavage of amyloid precursor protein (APP), are all shown to be mediated by metalloproteases. Reference is for example made to the reviews by Huovila et al., Trends Biochem Sci, (30), 7, 413-22 (2005); Seals and Courtneidge, Genes Dev 17, 7-30 (2003); Fox and Serrano, Toxicon 45, 969-85 (2005); Moss et al., Drug Discov Today, (6), 8, 417-426 (2001); Moss et al., Biochemistry, (43), 23, 7227-35 (2004); Handsley et al. Int J Cancer, (115), 6, 849-60 (2005); Herren, News Physiol Sci, (17), 73-6 (2002); and Hojilla et al., Br J Cancer, (89), 10, 1817-21 (2003); as well as to the further references cited therein and the further prior art cited in the present specification. Reference is for example made to FIG. 1 in the review by Huovila et al., supra; FIGS. 2 and 3 in Allinson et al., J Neurosci Res 74, 342-52 (2003); to FIGS. 1 and 3 in the review by Moss et al (2004), supra; to FIGS. 1 and 3 from the review by Seals and Courtneidge, supra; and to FIG. 1 in the review by Herren, supra.

The ADAM (A disintegrin And Metalloproteases) family includes proteins containing a disintegrin-like and metalloprotease-like domain). The ADAMTS contain a thrombospondin (TS) domain in addition to the other common domains (and should be considered included within the term ADAM proteinase as used in the specification and claims). Together with the Snake Venom Metalloproteinases (SVMPs), ADAM and ADAM-TS form the reprolysin subfamily of metalloprotease. All family members share a common multi-domain structure minimally containing a Pro domain, a metalloprotease (-like) and a disintegrin-like domain. Some ADAM have additional domains like cystein-rich and a transmembrane and cytoplasmic domains. Reference is again made to FIG. 1 in the review by Huovila et al., supra; FIGS. 2 and 3 in Allinson et al., J Neurosci Res 74, 342-52 (2003); to FIGS. 1 and 3 in the review by Moss et al (2004), supra; to FIGS. 1 and 3 from the review by Seals and Courtneidge, supra; and to FIG. 1 in the review by Herren, supra.

The substrates of ADAM metalloproteinases are also well-known, and for example include CD23, TNFR1, IL1-RII, CX3CL1, TNF-alpha, TNFR1, TNFRII, and TRANCE, as well as the substrates mentioned in Table 1 of the review by Huovila et al., supra.; and Table 1 in the review by Moss et al (2004), supra.

Members of the ADAM family are also well-known in the art, and for example and without limitation include:

-   -   ADAM1, ADAM2, ADAM3B, ADAM4, ADAM5, ADAM6, ADAM7, ADAM8, ADAM9,         ADAM10, ADAM11, ADAM12, ADAM13, ADAM14, ADAM15, ADAM16, ADAM17,         ADAM18, ADAM19, ADAM20, ADAM21, ADAM21, ADAM22, ADAM23, ADAM24,         ADAM25, ADAM26, ADAM27, ADAM28, ADAM29, ADAM30, ADAM31, ADAM32,         ADAM33, ADAM34, ADAM35, ADAM36, ADAM37, ADAM38, ADAM39, ADAM40     -   ADAMTS1, ADAMTS2, ADAMTS3, ADAMTS4, ADAMTS5, ADAMTS6, ADAMTS7,         ADAMTS8, ADAMTS9, ADAMTS10, ADAMTS11, ADAMTS12, ADAMTS 13,         ADAMTS 14, ADAMTS15, ADAMTS 16, ADAMTS 17, ADAMTS 18, ADAMTS 19,         ADAMTS20

Reference is also made to the following websites: http://merops.sanger.ac.uk/ and http://www.uta.fi/%7Eloiika/ADAMs/HADAMs.htm.

Diseases and disorders in which ADAM metalloproteinases are involved as also well-known in the art. Reference is again made to the reviews mentioned above and the further references cited therein; and also to for example to Levy et al., Nature 413, 488-94 (2001); Stanton et al.; Nature 434, 648-52 (2005); Glasson et al., Nature 434, 644-8 (2005), Weskamp et al., Nat Immunol 7, 1293-1298 (2006); Allinson et al., J Neurosci Res 74, 342-52 (2003); and Blobel, Nat Rev Mol Cell Biol 6, 32-43 (2005); and for example include:

-   -   ADAM8: allergy, inflammation (asthma, TRAPS)     -   ADAM9: hematological malignancies     -   ADAM10: chemotaxis, inflammation (asthma, rhinitis) Alzheimer     -   ADAM17/TACE: inflammation, IL1beta signaling, sepsis,         inflammatory, arthritis, diabetes, HIV cachexia, cancer and         TNF/EGF dependent diseases     -   ADAM-TS5: aggrecanase 2 and osteoarthritis, arthritis     -   ADAM15: atherosclerosis     -   ADAM33: Asthma

For example, ADAM17 is the major physiological TNFalpha converting enzyme and is therefore essential to TNFalpha signaling. Therefore, inhibition of ADAM17 will be useful in any disease state where TNF antagonist has been validated, such as arthritis, diabetes, HIV cachexia, sepsis and cancer. Reference is for example made to the review articles cited above and the further references cited herein and in the present specification. Moss et al. (2001) and Moss et al. (2004).

Also ADAM17 is responsible for the processing and release of TGFalpha and EGF. Indeed ADAM17−/− mice are embryonic lethal with phenotype reminiscent of the TGFalpha−/− and EGFR−/− mice. “Key functions of ADAMs have emerged in ErbB signalling pathways as being sheddases for multiple ErbB ligands. As the ErbB pathway is a validated target for anti-cancer drugs, the upstream activators of ErbB ligands, their sheddases, now enter the spotlight as new drug targets in the ErbB pathway. ADAMs are involved not only in tumour cell proliferation but also in angiogenesis and metastasis. Therefore, strategies targeting ADAMs might be an important complement to existing anti-ErbB approaches.” (See Blobel, supra).

ADAM10 and ADAM17 have been described as important sheddases for cytokines and their receptors, suggesting that these ADAMs are key modulator of cytokines in vivo.

The gene ADAM33, was identified as an asthma susceptibility gene involved in airway remodeling.

Increasing evidence point that ADAM10 (and to lesser extend ADAM17) is an alpha-secretase of the amyloid precursor protein (APP). Because cleavage of APP at the alpha site is believed to preclude cleavage at the beta and gamma site (cleavage sites promoting the APP aggregation leading to Alzheimer disease), ADAM10 can be considered as a protective factor in the etiology of Alzheimer's disease. Activation of ADAM10 might prove useful for the treatment of Alzheimer's disease (see for review Allinson 2003 and ref therein).

ADAM12 cleaves and inactivate IGFBP3, a natural inhibitor of IGF1 and 2. Because low level of IGF are associated with osteoarthritis and diabetes, the balance between IGFs and IGFBPs is crucial. Interestingly, mice overexpressing ADAM12 develop more abdominal and body fat. Also ADAM12−/− mice have reduced adipocytes and partial defect in myogenesis.

ADAM9 is expressed in hematological malignancies and might process the EGFR ligand HB-EGF.

ADAM8 is a serological and histochemical marker for lung cancer (Ishikawa N, 2004). ADAM8 may be the CD23 shedding enzyme. Soluble CD23 released from cells during allergic response stimulate IgE production. Specific inhibition of CD23 sheddase activity would be an important treatment of allergic reactions.

ADAM8 is (over)expressed in tissues around aseptically loosened total hip implants, which are characterized by chronic foreign body inflammation and peri-implant bone loss. This is compatible with a role for ADAM8 in the formation of foreign body giant cells and osteoclasts.

Higher ADAM19 expression has been associated with clinical and structural deterioration involved in renal disease, and ADAM19 may have a role in the dysfunctional renal allograft state.

In addition, expression levels and activities ADAM8 and ADAM19 are associated with invasiveness in human primary brain tumors.

Snake venom metalloproteinases play an important role in viperid envenoming (hemorrhage, edema, hypertension, hypovolemia, inflammation and necrosis) (see for example Fox et al., supra).

In mice, ADAM-TS5 is the major aggrecanase (aggrecan is a major component of the cartilage extracellular matrix) responsible of osteoarthritis and inflammatory arthritis.

ADAMTS-13 deficiency gives rise to thrombotic thrombocytopenic purpura (TTP), a life threatening condition (see for example Levy et al., supra)

Also, of course, modulation of ADAMs may have an influence on the biological pathways, physiological effects, signaling mechanisms and other biological and physiological activities that their respective substrates (and the ligands of such substrates) are involved in. Thus, it is expected that modulation of ADAMs could also play a part in the prevention and treatment of diseases and disorders in which their substrates are involved.

Another class of metalloproteinases closely related to the ADAM family are the snake venom metalloproteinases such as, without limitation, Aclpref, Acostatin, Acurhagin, Acutolysin A, Atrolysin, Atrolysin A, Atrolysin B, Atrolysin C, Atrolysin E, Atroxase, BAP1, Berythractivase, Bilitoxin-I, Bothropasin, Brevilysin H6, Catrocollastatin, Contortrostatin, Ecarin, Fibrolase, Flavoridin, Flavostatin, Graminelysin I, H2-Proteinase, HF3, HR1A, HR1B, HR2A, HT-2, HV1, Jararhagin, Jerdonitin, Kaouthiagin, Lebatase, LHF-II, MT-D, RVV-X, Trigramin, VAP1, VLFXA (see Fox et al., Toxicon 45 (2005), 969-985). Amino acid sequences and polypeptides of the invention could also be developed as antidotes for such venoms.

The polypeptides and compositions of the present invention can generally be used to modulate, and in particular inhibit and/or prevent, the interaction between a metalloproteinase from the ADAM family and its substrate (and in particular ADAM-mediated ectodomain-shedding, i.e. the ADAM-mediated release of extracellular domains from the substrate) and thus to modulate, and in particular inhibit or prevent, the signalling that is mediated by a metalloproteinase from the ADAM family and/or by its substrate, to modulate the biological pathways in which a metalloproteinase from the ADAM family and/or its substrate is involved, and/or to modulate the biological mechanisms, responses and effects associated with such signalling or these pathways.

As such, the polypeptides and compositions of the present invention can be used for the prevention and treatment (as defined herein) of ADAM-related diseases and disorders. Generally, “ADAM-related diseases and disorders” can be defined as diseases and disorders that can be prevented and/or treated, respectively, by suitably administering to a subject in need thereof (i.e. having the disease or disorder or at least one symptom thereof and/or at risk of attracting or developing the disease or disorder) of either a polypeptide or composition of the invention (and in particular, of a pharmaceutically active amount thereof) and/or of a known active principle active against a metalloproteinase from the ADAM family (or against one of its substrates, and/or against one of the ligands of such a substrate) or a biological pathway or mechanism in which a metalloproteinase from the ADAM family (and/or one of its substrates) is involved (and in particular, of a pharmaceutically active amount thereof). Examples of such ADAM-related diseases and disorders will be clear to the skilled person based on the disclosure herein, and for example include the diseases and disorders mentioned herein and in the prior art cited herein.

In particular, the polypeptides and compositions of the present invention can be used for the prevention and treatment of ADAM-related diseases and disorders which are characterized by excessive and/or unwanted signalling mediated by a substrate of a metalloproteinase from the ADAM family (or one of the ligands of such a substrate) or by the pathway(s) in which a metalloproteinase from the ADAM family is involved. Examples of such ADAM-related diseases and disorders will again be clear to the skilled person based on the disclosure herein.

Thus, without being limited thereto, the amino acid sequences and polypeptides of the invention can for example be used to prevent and/or to treat all diseases and disorders that are currently being prevented or treated with active principles that can modulate a metalloproteinase from the ADAM family-mediated signalling, such as those mentioned in the prior art cited above. It is also envisaged that the polypeptides of the invention can be used to prevent and/or to treat all diseases and disorders for which treatment with such active principles is currently being developed, has been proposed, or will be proposed or developed in future. In addition, it is envisaged that, because of their favourable properties as further described herein, the polypeptides of the present invention may be used for the prevention and treatment of other diseases and disorders than those for which these known active principles are being used or will be proposed or developed; and/or that the polypeptides of the present invention may provide new methods and regimens for treating the diseases and disorders described herein.

Other applications and uses of the amino acid sequences and polypeptides of the invention will become clear to the skilled person from the further disclosure herein.

Generally, it is an object of the invention to provide pharmacologically active agents, as well as compositions comprising the same, that can be used in the diagnosis, prevention and/or treatment of ADAM-related diseases and disorders and of the further diseases and disorders mentioned herein; and to provide methods for the diagnosis, prevention and/or treatment of such diseases and disorders that involve the administration and/or use of such agents and compositions.

In particular, it is an object of the invention to provide such pharmacologically active agents, compositions and/or methods that have certain advantages compared to the agents, compositions and/or methods that are currently used and/or known in the art. These advantages will become clear from the further description below.

More in particular, it is an object of the invention to provide therapeutic proteins that can be used as pharmacologically active agents, as well as compositions comprising the same, for the diagnosis, prevention and/or treatment of ADAM-related diseases and disorders and of the further diseases and disorders mentioned herein; and to provide methods for the diagnosis, prevention and/or treatment of such diseases and disorders that involve the administration and/or the use of such therapeutic proteins and compositions.

Accordingly, it is a specific object of the present invention to provide amino acid sequences that are directed against (as defined herein) a metalloproteinase from the ADAM family, in particular against a metalloproteinase from the ADAM family from a warm-blooded animal, more in particular against a metalloproteinase from the ADAM family from a mammal, and especially against human a metalloproteinase from the ADAM family; and to provide proteins and polypeptides comprising or essentially consisting of at least one such amino acid sequence.

In particular, it is a specific object of the present invention to provide such amino acid sequences and such proteins and/or polypeptides that are suitable for prophylactic, therapeutic and/or diagnostic use in a warm-blooded animal, and in particular in a mammal, and more in particular in a human being.

More in particular, it is a specific object of the present invention to provide such amino acid sequences and such proteins and/or polypeptides that can be used for the prevention, treatment, alleviation and/or diagnosis of one or more diseases, disorders or conditions associated with a metalloproteinase from the ADAM family and/or mediated by a metalloproteinase from the ADAM family (such as the diseases, disorders and conditions mentioned herein) in a warm-blooded animal, in particular in a mammal, and more in particular in a human being.

It is also a specific object of the invention to provide such amino acid sequences and such proteins and/or polypeptides that can be used in the preparation of pharmaceutical or veterinary compositions for the prevention and/or treatment of one or more diseases, disorders or conditions associated with and/or mediated by a metalloproteinase from the ADAM family (such as the diseases, disorders and conditions mentioned herein) in a warm-blooded animal, in particular in a mammal, and more in particular in a human being.

In the invention, generally, these objects are achieved by the use of the amino acid sequences, proteins, polypeptides and compositions that are described herein.

In general, the invention provides amino acid sequences that are directed against (as defined herein) and/or can specifically bind (as defined herein) to a metalloproteinase from the ADAM family; as well as compounds and constructs, and in particular proteins and polypeptides, that comprise at least one such amino acid sequence.

More in particular, the invention provides amino acid sequences that can bind to a metalloproteinase from the ADAM family with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein; as well as compounds and constructs, and in particular proteins and polypeptides, that comprise at least one such amino acid sequence.

In particular, amino acid sequences and polypeptides of the invention are preferably such that they:

-   -   bind to a metalloproteinase from the ADAM family with a         dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or         less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more         preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association         constant (K_(A)) of 10⁵ to 10¹² liter/ moles or more, and         preferably 10⁷ to 10¹² liter/moles or more and more preferably         10⁸ to 10¹² liter/moles);         and/or such that they:     -   bind to a metalloproteinase from the ADAM family with a         k_(on)-rate of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹,         preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably         between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹         and 10⁷ M⁻¹s⁻¹;         and/or such that they:     -   bind to a metalloproteinase from the ADAM family with a         k_(off)-rate between 1 s^(−1 (t) _(1/2)=0.69 s) and 10⁻⁶ s⁻¹         (providing a near irreversible complex with a t_(1/2) of         multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more         preferably between 10⁻³ s⁻¹ and 10⁻⁴ s⁻¹, such as between 10⁻⁴         s⁻¹ and 10⁻⁻⁶ s⁻¹.

Preferably, a monovalent amino acid sequence of the invention (or a polypeptide that contains only one amino acid sequence of the invention) is preferably such that it will bind to a metalloproteinase from the ADAM family with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

Some preferred IC50 values for binding of the amino acid sequences or polypeptides of the invention to a metalloproteinase from the ADAM family will become clear from the further description and examples herein.

For binding to a metalloproteinase from the ADAM family, an amino acid sequence of the invention will usually contain within its amino acid sequence one or more amino acid residues or one or more stretches of amino acid residues (i.e. with each “stretch” comprising two or amino acid residues that are adjacent to each other or in close proximity to each other, i.e. in the primary or tertiary structure of the amino acid sequence) via which the amino acid sequence of the invention can bind to a metalloproteinase from the ADAM family, which amino acid residues or stretches of amino acid residues thus form the “site” for binding to a metalloproteinase from the ADAM family (also referred to herein as the “antigen binding site”).

The amino acid sequences provided by the invention are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the invention (as defined herein), which may comprise or essentially consist of one or more amino acid sequences of the invention and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more amino acid sequences of the invention may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e. against one or more other targets than a metalloproteinase from the ADAM family), so as to provide a monovalent, multivalent or multispecific polypeptide of the invention, respectively, all as described herein. Such a protein or polypeptide may also be in essentially isolated form (as defined herein).

The amino acid sequences and polypeptides of the invention as such preferably essentially consist of a single amino acid chain that is not linked via disulphide bridges to any other amino acid sequence or chain (but that may or may not contain one or more intramolecular disulphide bridges. For example, it is known that Nanobodies—as described herein—may sometimes contain a disulphide bridge between CDR3 and CDR1 or FR2). However, it should be noted that one or more amino acid sequences of the invention may be linked to each other and/or to other amino acid sequences (e.g. via disulphide bridges) to provide peptide constructs that may also be useful in the invention (for example Fab′ fragments, F(ab′)₂ fragments, ScFv constructs, “diabodies” and other multispecific constructs. Reference is for example made to the review by Holliger and Hudson, Nat Biotechnol. 2005 September; 23(9):1126-36).

Generally, when an amino acid sequence of the invention (or a compound, construct or polypeptide comprising the same) is intended for administration to a subject (for example for therapeutic and/or diagnostic purposes as described herein), it is preferably either an amino acid sequence that does not occur naturally in said subject; or, when it does occur naturally in said subject, in essentially isolated form (as defined herein).

It will also be clear to the skilled person that for pharmaceutical use, the amino acid sequences of the invention (as well as compounds, constructs and polypeptides comprising the same) are preferably directed against human a metalloproteinase from the ADAM family; whereas for veterinary purposes, the amino acid sequences and polypeptides of the invention are preferably directed against a metalloproteinase from the ADAM family from the species to be treated, or at least cross-reactive with a metalloproteinase from the ADAM family from the species to be treated.

Furthermore, an amino acid sequence of the invention may optionally, and in addition to the at least one binding site for binding against a metalloproteinase from the ADAM family, contain one or more further binding sites for binding against other antigens, proteins or targets.

The efficacy of the amino acid sequences and polypeptides of the invention, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease or disorder involved. Suitable assays and animal models will be clear to the skilled person, and for example include techniques such as BIACORE and FLIPR; commercially available protease assays (for example the assays for ADAM-(TS) available from R&D Systems. For ADAM8, 9, 10 and 17 the ES003 substrate can be used); cell-based assays that measure sheddase activity (for example for CD23, EGF or TNF, see Weskamp et at, supra and the references cited therein), as well as the assays and animal models used in the experimental part below and in the prior art cited herein. Also, assays can be used that measure the activity of the substrate of ADAM or the signalling or biological activities in which such a substrate (or its natural ligand) in involved.

For example, TNFalpha release (cleavage) can be induced in monocytes and inactivating or activating ADAM17 modulators can be tested in this setup. These and similar assays are for example described in Newton et al. Ann Rheum Dis 60 Suppl 3, iii25-32 (2001).

In animal models, ADAM10 activity can be quantified using soluble CD23 (see for example Weskamp et at 2006, supra, and the references cited therein). Antigen induced arthritis model can be used to test ADAM-TS5 Nanobodies. (ADAM-TS5−/− mice are more resistant to arthritis in this model).

An ELISA-based kit can be purchased from Invitek to test the activity of amino acid sequences or polypeptides of the invention on ADAM-TS5. For testing the activity of of amino acid sequences or polypeptides of the invention on ADAM10 in a cell-based assay, cleavage of CD44 into the medium of cell can be used to detect the activity of ADAM10 in U251 glioblastoma (S. Atkinson and G. Murphy, publication submitted). For testing the activity of of amino acid sequences or polypeptides of the invention on ADAM17 in a cell-based assay, the release of HB-EGF conjugated Alkaline phosphatase from transfected MCF7 cells can be used a readout for ADAM17 activity (S. Atkinson and G. Murphy, submitted).

Also, according to the invention, amino acid sequences and polypeptides that are directed against a metalloproteinase from the ADAM family from a first species of warm-blooded animal may or may not show cross-reactivity with a metalloproteinase from the ADAM family from one or more other species of warm-blooded animal. For example, amino acid sequences and polypeptides directed against human a metalloproteinase from the ADAM family may or may not show cross reactivity with a metalloproteinase from the ADAM family from one or more other species of primates (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus)) and/or with a metalloproteinase from the ADAM family from one or more species of animals that are often used in animal models for diseases (for example mouse, rat, rabbit, pig or dog), and in particular in animal models for diseases and disorders associated with a metalloproteinase from the ADAM family (such as the species and animal models mentioned herein). In this respect, it will be clear to the skilled person that such cross-reactivity, when present, may have advantages from a drug development point of view, since it allows the amino acid sequences and polypeptides against human a metalloproteinase from the ADAM family to be tested in such disease models.

More generally, amino acid sequences and polypeptides of the invention that are cross-reactive with a metalloproteinase from the ADAM family from multiple species of mammal will usually be advantageous for use in veterinary applications, since it will allow the same amino acid sequence or polypeptide to be used across multiple species. Thus, it is also encompassed within the scope of the invention that amino acid sequences and polypeptides directed against a metalloproteinase from the ADAM family from one species of animal (such as amino acid sequences and polypeptides against human a metalloproteinase from the ADAM family) can be used in the treatment of another species of animal, as long as the use of the amino acid sequences and/or polypeptides provide the desired effects in the species to be treated.

The present invention is in its broadest sense also not particularly limited to or defined by a specific antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of a metalloproteinase from the ADAM family against which the amino acid sequences and polypeptides of the invention are directed. For example, the amino acid sequences and polypeptides may or may not be directed against an “interaction site” (as defined herein). However, some preferred amino acid sequences of the invention may be directed against one or more of the following epitopes, domains, active/catalytic sites or binding sites:

-   -   against the catalytic site: such amino acid sequences may for         example reduce, inhibit or inactivate the protease activity;     -   against the catalytic domain and/or the PRO-domain-catalytic         site (such that it is disrupted): such interaction may for         example activate or increase the protease activity;     -   against the PRO-domain and/or against the PRO-catalytic site         (such that it is disrupted): such interaction may for example         activate or increase the protease activity;     -   against the Disintegrin/cys-rich domain: such interaction may         for example modulate the targeting of the ADAM or its binding to         substrate, or its selectivity for the substrate;     -   against the site on the ADAM proteinase for interaction with         TIMP, thus modulating (and in particular inhibiting) the         regulation of ADAM by TIMPs (TIMP1,2,3 or 4).

Also, generally, some preferred amino acid sequences and polypeptides of the invention are preferably such that they are capable of inhibiting the activity of the ADAM proteinase, as measured by a suitable assay (such as a commercially available protease assay) under conditions usually applied for such an assay (as will be clear to the skilled person), by at least 1%, preferably at least 5%, more preferably at least 10%, such as at least 25% or even more than 50% and up to 75% or more, such as 90% or more, compared to the activity of the ADAM proteinase without the presence of the amino acid sequence or polypeptide of the invention.

Also, generally, some preferred amino acid sequences and polypeptides of the invention are preferably such that they are capable of activating (the protease activity of) the ADAM proteinase, as measured by a suitable assay (such as a commercially available protease assay) under conditions usually applied for such an assay (as will be clear to the skilled person), by at least 1%, preferably at least 5%, more preferably at least 10%, such as at least 25% or even more than 50% and up to 75% or more, such as 90% or more, compared to the activity of the ADAM proteinase without the presence of the amino acid sequence or polypeptide of the invention.

Some other preferred amino acid sequences and polypeptides may be such that they are capable of competing for binding to the ADAM proteinase with the usual substrate of the ADAM proteinase, and/or with a substrate for an ADAM proteinase that is usually used as a model substrate in an assay for determining the activity of said proteinase.

Some other preferred amino acid sequences and polypeptides may be such that they are capable of modulating (as defined herein) the regulation of the ADAM proteinase by TIMP. As is known in the art, therapies involving the use of TIMP or targeting may lead to an undesirable inhibition of metalloproteases, which may cause side effects. Thus, one aspect of the invention relates to the use of an amino acid sequence, Nanobody or polypeptide of the invention, or of a composition comprising the same (methods or compositions for) preventing or reducing the side effects of TIMP-dependent therapies or therapies directed against or mediated by TIMP; and to uses of the amino acid sequences, Nanobodies and polypeptides of the invention in the preparation of pharmaceutical compositions for reducing the side effects of TIMP-dependent therapies or therapies directed against or mediated by TIMP; or with such reduced side effects.

The invention also relates to the use of an amino acid sequence, Nanobody or polypeptide of the invention, or of a composition comprising the same, in (methods or compositions for) modulating (as defined herein) an ADAM metalloproteinase, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or in a multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from ADAM-related diseases and disorders).

The invention also relates to methods for modulating (as defined herein) an ADAM metalloproteinase, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from ADAM-related diseases and disorders), which method comprises at least the step of contacting an ADAM metalloproteinase with at least one amino acid sequence, Nanobody or polypeptide of the invention, or with a composition comprising the same, in a manner and in an amount suitable to modulate an ADAM metalloproteinase, with at least one amino acid sequence, Nanobody or polypeptide of the invention.

The invention also relates to the use of an one amino acid sequence, Nanobody or polypeptide of the invention in the preparation of a composition (such as, without limitation, a pharmaceutical composition or preparation as further described herein) for modulating (as defined herein) an ADAM metalloproteinase, either in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in an a single cell or multicellular organism, and in particular in a mammal, and more in particular in a human being, such as in a human being that is at risk of or suffers from a ADAM-related diseases and disorders).

The amino acid sequences, Nanobodies and polypeptides of the invention may also be in the prevention and treatment of sepsis. Thus, further aspects of the invention relate to the use of an amino acid sequence, Nanobody or polypeptide of the invention, or of a composition comprising the same, in (methods or compositions for) the treatment (as defined herein) of sepsis; to pharmaceutical compositions and preparations (as described herein) for the treatment of sepsis; and to the use of the amino acid sequences, Nanobodies and polypeptides of the invention in methods for preparing pharmaceutical compositions and preparations (as described herein) for the treatment of sepsis.

The amino acid sequences, Nanobodies and polypeptides of the invention may also be used in the prevention and treatment of the side effects of TIMP-related therapies. Thus, further aspects of the invention relate to the use of an amino acid sequence, Nanobody or polypeptide of the invention, or of a composition comprising the same, in (methods or compositions for) the prevention and/or treatment (as defined herein) of the side effects of TIMP-related therapies; to pharmaceutical compositions and preparations (as described herein) for the prevention and/or treatment (as defined herein) of the side effects of TIMP-related therapies; and to the use of the amino acid sequences, Nanobodies and polypeptides of the invention in methods for preparing pharmaceutical compositions and preparations (as described herein) for the prevention and/or treatment (as defined herein) of the side effects of TIMP-related therapies.

The amino acid sequences, Nanobodies and polypeptides of the invention may also be used to inhibit or reduce the release of one or more growth factors, for example in the treatment of cancer. Thus, further aspects of the invention relate to the use of an amino acid sequence, Nanobody or polypeptide of the invention, or of a composition comprising the same, in (methods or compositions for) inhibiting or reducing the release of one or more growth factors, for example in the treatment of cancer; to pharmaceutical compositions and preparations (as described herein) for inhibiting or reducing the release of one or more growth factors, for example in the treatment of cancer; and to the use of the amino acid sequences, Nanobodies and polypeptides of the invention in methods for preparing pharmaceutical compositions and preparations (as described herein) for inhibiting or reducing the release of one or more growth factors, for example in the treatment of cancer.

The amino acid sequences, Nanobodies and polypeptides of the invention may also be used to inhibit or reduce the release of one or more cytokines, for example in the prevention or treatment of inflammation. Thus, further aspects of the invention relate to the use of an amino acid sequence, Nanobody or polypeptide of the invention, or of a composition comprising the same, in (methods or compositions for) inhibiting or reducing the release of one or more cytokines, for example in the prevention or treatment of inflammation; to pharmaceutical compositions and preparations (as described herein) for inhibiting or reducing the release of one or more cytokines, for example in the prevention or treatment of inflammation; and to the use of the amino acid sequences, Nanobodies and polypeptides of the invention in methods for preparing pharmaceutical compositions and preparations (as described herein) for inhibiting or reducing the release of one or more cytokines, for example in the prevention or treatment of inflammation.

The amino acid sequences, Nanobodies and polypeptides of the invention may also be used as a marker for cells that express ADAM proteinases (such as ADAM15) on their surface, for example for detecting tumour cells or atherosclerosis, for example in vitro or by means of in vivo imaging. Reference is made to the further disclosure herein.

As further described herein, a polypeptide of the invention may contain two or more amino acid sequences of the invention that are directed against an ADAM proteinase. Generally, such polypeptides will bind to the ADAM proteinase with increased avidity compared to a single amino acid sequence of the invention. Such a polypeptide may for example comprise two amino acid sequences of the invention that are directed against the same antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of ADAM proteinase (which may or may not be an interaction site); or comprise at least one “first” amino acid sequence of the invention that is directed against a first same antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of ADAM proteinase (which may or may not be an interaction site); and at least one “second” amino acid sequence of the invention that is directed against a second antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) different from the first (and which again may or may not be an interaction site). Preferably, in such “biparatopic” polypeptides of the invention, at least one amino acid sequence of the invention is directed against an interaction site (as defined herein), although the invention in its broadest sense is not limited thereto.

Also, when the target is part of a binding pair (for example, a receptor-ligand binding pair), the amino acid sequences and polypeptides may be such that they compete with the cognate binding partner (e.g. the ligand, receptor or other binding partner, as applicable) for binding to the target, and/or such that they (fully or partially) neutralize binding of the binding partner to the target.

It is also within the scope of the invention that, where applicable, an amino acid sequence of the invention can bind to two or more antigenic determinants, epitopes, parts, domains, subunits or confirmations of a metalloproteinase from the ADAM family. In such a case, the antigenic determinants, epitopes, parts, domains or subunits of a metalloproteinase from the ADAM family to which the amino acid sequences and/or polypeptides of the invention bind may be essentially the same (for example, if a metalloproteinase from the ADAM family contains repeated structural motifs or occurs in a multimeric form) or may be different (and in the latter case, the amino acid sequences and polypeptides of the invention may bind to such different antigenic determinants, epitopes, parts, domains, subunits of a metalloproteinase from the ADAM family with an affinity and/or specificity which may be the same or different). Also, for example, when a metalloproteinase from the ADAM family exists in an activated conformation and in an inactive conformation, the amino acid sequences and polypeptides of the invention may bind to either one of these confirmation, or may bind to both these confirmations (i.e. with an affinity and/or specificity which may be the same or different). Also, for example, the amino acid sequences and polypeptides of the invention may bind to a conformation of a metalloproteinase from the ADAM family in which it is bound to a pertinent ligand, may bind to a conformation of a metalloproteinase from the ADAM family in which it not bound to a pertinent ligand, or may bind to both such conformations (again with an affinity and/or specificity which may be the same or different).

It is also expected that the amino acid sequences and polypeptides of the invention will generally bind to all naturally occurring or synthetic analogs, variants, mutants, alleles, parts and fragments of a metalloproteinase from the ADAM family; or at least to those analogs, variants, mutants, alleles, parts and fragments of a metalloproteinase from the ADAM family that contain one or more antigenic determinants or epitopes that are essentially the same as the antigenic determinant(s) or epitope(s) to which the amino acid sequences and polypeptides of the invention bind in a metalloproteinase from the ADAM family (e.g. in wild-type a metalloproteinase from the ADAM family). Again, in such a case, the amino acid sequences and polypeptides of the invention may bind to such analogs, variants, mutants, alleles, parts and fragments with an affinity and/or specificity that are the same as, or that are different from (i.e. higher than or lower than), the affinity and specificity with which the amino acid sequences of the invention bind to (wild-type) a metalloproteinase from the ADAM family. It is also included within the scope of the invention that the amino acid sequences and polypeptides of the invention bind to some analogs, variants, mutants, alleles, parts and fragments of a metalloproteinase from the ADAM family, but not to others.

When a metalloproteinase from the ADAM family exists in a monomeric form and in one or more multimeric forms, it is within the scope of the invention that the amino acid sequences and polypeptides of the invention only bind to a metalloproteinase from the ADAM family in monomeric form, only bind to a metalloproteinase from the ADAM family in multimeric form, or bind to both the monomeric and the multimeric form. Again, in such a case, the amino acid sequences and polypeptides of the invention may bind to the monomeric form with an affinity and/or specificity that are the same as, or that are different from (i.e. higher than or lower than), the affinity and specificity with which the amino acid sequences of the invention bind to the multimeric form.

Also, when a metalloproteinase from the ADAM family can associate with other proteins or polypeptides to form protein complexes (e.g. with multiple subunits), it is within the scope of the invention that the amino acid sequences and polypeptides of the invention bind to a metalloproteinase from the ADAM family in its non-associated state, bind to a metalloproteinase from the ADAM family in its associated state, or bind to both. In all these cases, the amino acid sequences and polypeptides of the invention may bind to such multimers or associated protein complexes with an affinity and/or specificity that may be the same as or different from (i.e. higher than or lower than) the affinity and/or specificity with which the amino acid sequences and polypeptides of the invention bind to a metalloproteinase from the ADAM family in its monomeric and non-associated state.

Also, as will be clear to the skilled person, proteins or polypeptides that contain two or more amino acid sequences directed against a metalloproteinase from the ADAM family may bind with higher avidity to a metalloproteinase from the ADAM family than the corresponding monomeric amino acid sequence(s). For example, and without limitation, proteins or polypeptides that contain two or more amino acid sequences directed against different epitopes of a metalloproteinase from the ADAM family may (and usually will) bind with higher avidity than each of the different monomers, and proteins or polypeptides that contain two or more amino acid sequences directed against a metalloproteinase from the ADAM family may (and usually will) bind also with higher avidity to a multimer of a metalloproteinase from the ADAM family.

Generally, amino acid sequences and polypeptides of the invention will at least bind to those forms of a metalloproteinase from the ADAM family (including monomeric, multimeric and associated forms) that are the most relevant from a biological and/or therapeutic point of view, as will be clear to the skilled person.

It is also within the scope of the invention to use parts, fragments, analogs, mutants, variants, alleles and/or derivatives of the amino acid sequences and polypeptides of the invention, and/or to use proteins or polypeptides comprising or essentially consisting of one or more of such parts, fragments, analogs, mutants, variants, alleles and/or derivatives, as long as these are suitable for the uses envisaged herein. Such parts, fragments, analogs, mutants, variants, alleles and/or derivatives will usually contain (at least part of) a functional antigen-binding site for binding against a metalloproteinase from the ADAM family; and more preferably will be capable of specific binding to a metalloproteinase from the ADAM family, and even more preferably capable of binding to a metalloproteinase from the ADAM family with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein. Some non-limiting examples of such parts, fragments, analogs, mutants, variants, alleles, derivatives, proteins and/or polypeptides will become clear from the further description herein. Additional fragments or polypeptides of the invention may also be provided by suitably combining (i.e. by linking or genetic fusion) one or more (smaller) parts or fragments as described herein.

In one specific, but non-limiting aspect of the invention, which will be further described herein, such analogs, mutants, variants, alleles, derivatives have an increased half-life in serum (as further described herein) compared to the amino acid sequence from which they have been derived. For example, an amino acid sequence of the invention may be linked (chemically or otherwise) to one or more groups or moieties that extend the half-life (such as PEG), so as to provide a derivative of an amino acid sequence of the invention with increased half-life.

In one specific, but non-limiting aspect, the amino acid sequence of the invention may be an amino acid sequence that comprises an immunoglobulin fold or may be an amino acid sequence that, under suitable conditions (such as physiological conditions) is capable of forming an immunoglobulin fold (i.e. by folding). Reference is inter alia made to the review by Halaby et al., J. (1999) Protein Eng. 12, 563-71. Preferably, when properly folded so as to form an immunoglobulin fold, such an amino acid sequence is capable of specific binding (as defined herein) to a metalloproteinase from the ADAM family; and more preferably capable of binding to a metalloproteinase from the ADAM family with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein. Also, parts, fragments, analogs, mutants, variants, alleles and/or derivatives of such amino acid sequences are preferably such that they comprise an immunoglobulin fold or are capable for forming, under suitable conditions, an immunoglobulin fold.

In particular, but without limitation, the amino acid sequences of the invention may be amino acid sequences that essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively); or any suitable fragment of such an amino acid sequence (which will then usually contain at least some of the amino acid residues that form at least one of the CDR's, as further described herein).

The amino acid sequences of the invention may in particular be an immunoglobulin sequence or a suitable fragment thereof, and more in particular be an immunoglobulin variable domain sequence or a suitable fragment thereof, such as light chain variable domain sequence (e.g. a V_(L)-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g. a V_(H)-sequence) or a suitable fragment thereof. When the amino acid sequence of the invention is a heavy chain variable domain sequence, it may be a heavy chain variable domain sequence that is derived from a conventional four-chain antibody (such as, without limitation, a V_(H) sequence that is derived from a human antibody) or be a so-called V_(HH)-sequence (as defined herein) that is derived from a so-called “heavy chain antibody” (as defined herein).

However, it should be noted that the invention is not limited as to the origin of the amino acid sequence of the invention (or of the nucleotide sequence of the invention used to express it), nor as to the way that the amino acid sequence or nucleotide sequence of the invention is (or has been) generated or obtained. Thus, the amino acid sequences of the invention may be naturally occurring amino acid sequences (from any suitable species) or synthetic or semi-synthetic amino acid sequences. In a specific but non-limiting aspect of the invention, the amino acid sequence is a naturally occurring immunoglobulin sequence (from any suitable species) or a synthetic or semi-synthetic immunoglobulin sequence, including but not limited to “humanized” (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized V_(HH) sequences or Nanobodies), “camelized” (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing. Reference is for example made to the standard handbooks, as well as to the further description and prior art mentioned herein.

Similarly, the nucleotide sequences of the invention may be naturally occurring nucleotide sequences or synthetic or semi-synthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), nucleotide sequence that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.

The amino acid sequence of the invention may in particular be a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody), a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a “dAb” (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody™ (as defined herein, and including but not limited to a V_(HH) sequence); other single variable domains, or any suitable fragment of any one thereof. For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684. For the term “dAb's”, reference is for example made to Ward et al. (Nature 1989 Oct. 12; 341 (6242): 544-6), to Holt et al., Trends Biotechnol., 2003, 21(11):484-490; as well as to for example WO 06/030220, WO 06/003388 and other published patent applications of Domantis Ltd. It should also be noted that, although less preferred in the context of the present invention because they are not of mammalian origin, single domain antibodies or single variable domains can be derived from certain species of shark (for example, the so-called “IgNAR domains”, see for example WO 05/18629).

In particular, the amino acid sequence of the invention may be a Nanobody™ (as defined herein) or a suitable fragment thereof. [Note: Nanobody®, Nanobodies® and Nanoclone® are registered trademarks of Ablynx N.V.] Such Nanobodies directed against a metalloproteinase from the ADAM family will also be referred to herein as “Nanobodies of the invention”.

For a general description of Nanobodies, reference is made to the further description below, as well as to the prior art cited herein. In this respect, it should however be noted that this description and the prior art mainly described Nanobodies of the so-called “V_(H)3 class” (i.e. Nanobodies with a high degree of sequence homology to human germline sequences of the V_(H)3 class such as DP-47, DP-51 or DP-29), which Nanobodies form a preferred aspect of this invention. It should however be noted that the invention in its broadest sense generally covers any type of Nanobody directed against a metalloproteinase from the ADAM family, and for example also covers the Nanobodies belonging to the so-called “V_(H)4 class” (i.e. Nanobodies with a high degree of sequence homology to human germline sequences of the V_(H)4 class such as DP-78), as for example described in the U.S. provisional application 60/792,279 by Ablynx N.V. entitled “DP-78-like Nanobodies” filed on Apr. 14, 2006 (see also PCT/EP2007/003259).

Generally, Nanobodies (in particular V_(HH) sequences and partially humanized Nanobodies) can in particular be characterized by the presence of one or more “Hallmark residues” (as described herein) in one or more of the framework sequences (again as further described herein).

Thus, generally, a Nanobody can be defined as an amino acid sequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which one or more of the Hallmark residues are as further         defined herein.

In particular, a Nanobody can be an amino acid sequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which the framework sequences are as further defined herein.

More in particular, a Nanobody can be an amino acid sequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which:

-   a) preferably one or more of the amino acid residues at positions     11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat     numbering are chosen from the Hallmark residues mentioned in Table     A-3 below;     and in which:

-   b) said amino acid sequence has at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 1 to     22, in which for the purposes of determining the degree of amino     acid identity, the amino acid residues that form the CDR sequences     (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are     disregarded.

In these Nanobodies, the CDR sequences are generally as further defined herein.

Thus, the invention also relates to such Nanobodies that can bind to (as defined herein) and/or are directed against a metalloproteinase from the ADAM family, to suitable fragments thereof, as well as to polypeptides that comprise or essentially consist of one or more of such Nanobodies and/or suitable fragments.

SEQ ID NO's 868 to 973 and 1044 to 1053 give the amino acid sequences of a number of V_(HH) sequences that have been raised against a metalloproteinase from the ADAM family, i.e.:

-   -   SEQ ID NO's 868 to 886: Nanobodies against ADAM8     -   SEQ ID NO's 887 to 907 and 1044 to 1047: Nanobodies against         ADAM9     -   SEQ ID NO's 908 to 931: Nanobodies against ADAM10     -   SEQ ID NO's 932 to 948 and 1048 to 1052: Nanobodies against         ADAM17     -   SEQ ID NO's 949 to 974 and 1053: Nanobodies against ADAMTS5

In particular, the invention in some specific aspects provides:

-   -   amino acid sequences that are directed against (as defined         herein) ADAM8 and that have at least 80%, preferably at least         85%, such as 90% or 95% or more sequence identity with at least         one of the amino acid sequences of SEQ ID NO's: 868 to 886.         These amino acid sequences may further be such that they         neutralize binding of the cognate ligand to ADAM8; and/or         compete with the cognate ligand for binding to ADAM8; and/or are         directed against an interaction site (as defined herein) on         ADAM8 (such as the ligand binding site);     -   amino acid sequences that cross-block (as defined herein) the         binding of at least one of the amino acid sequences of SEQ ID         NO's: 868 to 886 to ADAM8 and/or that compete with at least one         of the amino acid sequences of SEQ ID NO's: 868 to 886 for         binding to ADAM8. Again, these amino acid sequences may further         be such that they neutralize binding of the cognate ligand to         ADAM8; and/or compete with the cognate ligand for binding to         ADAM8; and/or are directed against an interaction site (as         defined herein) on ADAM8 (such as the ligand binding site);     -   amino acid sequences that are directed against (as defined         herein) ADAM9 and that have at least 80%, preferably at least         85%, such as 90% or 95% or more sequence identity with at least         one of the amino acid sequences of SEQ ID NO's: 887 to 907 and         1044 to 1047. These amino acid sequences may further be such         that they neutralize binding of the cognate ligand to ADAM9;         and/or compete with the cognate ligand for binding to ADAM9;         and/or are directed against an interaction site (as defined         herein) on ADAM9 (such as the ligand binding site);     -   amino acid sequences that cross-block (as defined herein) the         binding of at least one of the amino acid sequences of SEQ ID         NO's: 887 to 907 and 1044 to 1047 to ADAM9 and/or that compete         with at least one of the amino acid sequences of SEQ ID NO's:         887 to 907 and 1044 to 1047 for binding to ADAM9. Again, these         amino acid sequences may further be such that they neutralize         binding of the cognate ligand to ADAM9; and/or compete with the         cognate ligand for binding to ADAM9; and/or are directed against         an interaction site (as defined herein) on ADAM9 (such as the         ligand binding site);     -   amino acid sequences that are directed against (as defined         herein) ADAM10 and that have at least 80%, preferably at least         85%, such as 90% or 95% or more sequence identity with at least         one of the amino acid sequences of SEQ ID NO's: 908 to 931.         These amino acid sequences may further be such that they         neutralize binding of the cognate ligand to ADAM10; and/or         compete with the cognate ligand for binding to ADAM10; and/or         are directed against an interaction site (as defined herein) on         ADAM10 (such as the ligand binding site);     -   amino acid sequences that cross-block (as defined herein) the         binding of at least one of the amino acid sequences of SEQ ID         NO's: 908 to 931 to ADAM10 and/or that compete with at least one         of the amino acid sequences of SEQ ID NO's: 908 to 931 for         binding to ADAM10. Again, these amino acid sequences may further         be such that they neutralize binding of the cognate ligand to         ADAM10; and/or compete with the cognate ligand for binding to         ADAM10; and/or are directed against an interaction site (as         defined herein) on ADAM10 (such as the ligand binding site);     -   amino acid sequences that are directed against (as defined         herein) ADAM17 and that have at least 80%, preferably at least         85%, such as 90% or 95% or more sequence identity with at least         one of the amino acid sequences of SEQ ID NO's: 932 to 948 and         1048 to 1052. These amino acid sequences may further be such         that they neutralize binding of the cognate ligand to ADAM17;         and/or compete with the cognate ligand for binding to ADAM17;         and/or are directed against an interaction site (as defined         herein) on ADAM 17 (such as the ligand binding site);     -   amino acid sequences that cross-block (as defined herein) the         binding of at least one of the amino acid sequences of SEQ ID         NO's: 932 to 948 and 1048 to 1052 to ADAM17 and/or that compete         with at least one of the amino acid sequences of SEQ ID NO's:         932 to 948 and 1048 to 1052 for binding to ADAM17. Again, these         amino acid sequences may further be such that they neutralize         binding of the cognate ligand to ADAM17; and/or compete with the         cognate ligand for binding to ADAM17; and/or are directed         against an interaction site (as defined herein) on ADAM 17 (such         as the ligand binding site);     -   amino acid sequences that are directed against (as defined         herein) ADAMTS5 and that have at least 80%, preferably at least         85%, such as 90% or 95% or more sequence identity with at least         one of the amino acid sequences of SEQ ID NO's: 949 to 974         and 1053. These amino acid sequences may further be such that         they neutralize binding of the cognate ligand to ADAMTS5; and/or         compete with the cognate ligand for binding to ADAMTS5; and/or         are directed against an interaction site (as defined herein) on         ADAMTS5 (such as the ligand binding site);     -   amino acid sequences that cross-block (as defined herein) the         binding of at least one of the amino acid sequences of SEQ ID         NO's: 949 to 974 and 1053 to ADAMTS5 and/or that compete with at         least one of the amino acid sequences of SEQ ID NO's: 949 to 974         and 1053 for binding to ADAMTS5. Again, these amino acid         sequences may further be such that they neutralize binding of         the cognate ligand to ADAMTS5; and/or compete with the cognate         ligand for binding to ADAMTS5; and/or are directed against an         interaction site (as defined herein) on ADAMTS5 (such as the         ligand binding site);         which amino acid sequences may be as further described herein         (and may for example be Nanobodies); as well as polypeptides of         the invention that comprise one or more of such amino acid         sequences (which may be as further described herein, and may for         example be bispecific and/or biparatopic polypeptides as         described herein), and nucleic acid sequences that encode such         amino acid sequences and polypeptides. Such amino acid sequences         and polypeptides do not include any naturally occurring ligands.

In some other specific aspects, the invention provides:

-   -   amino acid sequences of the invention that are specific for (as         defined herein) ADAM8 compared to ADAM9, ADAM10, ADAM17 and/or         ADAMTS5. These preferably have have at least 80%, preferably at         least 85%, such as 90% or 95% or more sequence identity with at         least one of the amino acid sequences of SEQ ID NO's: 868 to         886;     -   amino acid sequences of the invention that are specific for (as         defined herein) ADAM9 compared to ADAM8, ADAM10, ADAM17 and/or         ADAMTS5. These preferably have at least 80%, preferably at least         85%, such as 90% or 95% or more sequence identity with at least         one of the amino acid sequences of SEQ ID NO's: 887 to 907 and         1044 to 1047.     -   amino acid sequences of the invention that are specific for (as         defined herein) ADAM10 compared to ADAM8, ADAM9, ADAM17 and/or         ADAMTS5. These preferably have at least 80%, preferably at least         85%, such as 90% or 95% or more sequence identity with at least         one of the amino acid sequences of SEQ ID NO's: 908 to 931;     -   amino acid sequences of the invention that are specific for (as         defined herein) ADAM17 compared to ADAM8, ADAM9, ADAM10 and/or         ADAMTS5. These preferably have at least 80%, preferably at least         85%, such as 90% or 95% or more sequence identity with at least         one of the amino acid sequences of SEQ ID NO's: 932 to 948 and         1048 to 1052;     -   amino acid sequences of the invention that are specific for (as         defined herein) ADAMTS5 compared to ADAM8, ADAM9, ADAM10 and/or         ADAM17. These preferably have at least 80%, preferably at least         85%, such as 90% or 95% or more sequence identity with at least         one of the amino acid sequences of SEQ ID NO's: 949 to 974 and         1053;         which amino acid sequences of the invention may be as further         described herein (and may for example be Nanobodies); as well as         polypeptides of the invention that comprise one or more of such         amino acid sequences (which may be as further described herein,         and may for example be bispecific and/or biparatopic         polypeptides as described herein), and nucleic acid sequences         that encode such amino acid sequences and polypeptides. Such         amino acid sequences and polypeptides do not include any         naturally occurring ligands.

Accordingly, some particularly preferred Nanobodies of the invention are Nanobodies which can bind (as further defined herein) to and/or are directed against to a metalloproteinase from the ADAM family and which:

-   a) have at least 80% amino acid identity with at least one of the     amino acid sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053,     in which for the purposes of determining the degree of amino acid     identity, the amino acid residues that form the CDR sequences are     disregarded. In this respect, reference is also made to Table A-1,     which lists the framework 1 sequences (SEQ ID NO's: 126 to 231),     framework 2 sequences (SEQ ID NO's: 338 to 443), framework 3     sequences (SEQ ID NO's: 550 to 655) and framework 4 sequences (SEQ     ID NO's: 762 to 867) of the Nanobodies of SEQ ID NO's: 868 to 973     and/or 1044 to 1053 (with respect to the amino acid residues at     positions 1 to 4 and 27 to 30 of the framework 1 sequences,     reference is also made to the comments made below. Thus, for     determining the degree of amino acid identity, these residues are     preferably disregarded);     and in which: -   b) preferably one or more of the amino acid residues at positions     11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat     numbering are chosen from the Hallmark residues mentioned in Table     A-3 below.

In these Nanobodies, the CDR sequences are generally as further defined herein.

Again, such Nanobodies may be derived in any suitable manner and from any suitable source, and may for example be naturally occurring V_(HH) sequences (i.e. from a suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences, including but not limited to “humanized” (as defined herein) Nanobodies, “camelized” (as defined herein) immunoglobulin sequences (and in particular camelized heavy chain variable domain sequences), as well as Nanobodies that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing as further described herein. Also, when a Nanobody comprises a V_(HH) sequence, said Nanobody may be suitably humanized, as further described herein, so as to provide one or more further (partially or fully) humanized Nanobodies of the invention. Similarly, when a Nanobody comprises a synthetic or semi-synthetic sequence (such as a partially humanized sequence), said Nanobody may optionally be further suitably humanized, again as described herein, again so as to provide one or more further (partially or fully) humanized Nanobodies of the invention.

In particular, humanized Nanobodies may be amino acid sequences that are as generally defined for Nanobodies in the previous paragraphs, but in which at least one amino acid residue is present (and in particular, in at least one of the framework residues) that is and/or that corresponds to a humanizing substitution (as defined herein). Some preferred, but non-limiting humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein. In addition, or alternatively, other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring V_(HH) sequence with the corresponding framework sequence of one or more closely related human V_(H) sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said V_(HH) sequence (in any manner known per se, as further described herein) and the resulting humanized V_(HH) sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework regions of) a Nanobody may be partially humanized or fully humanized.

Some particularly preferred humanized Nanobodies of the invention are humanized variants of the Nanobodies of SEQ ID NO's: 868 to 973 and/or 1044 to 1053.

Thus, some other preferred Nanobodies of the invention are Nanobodies which can bind (as further defined herein) to a metalloproteinase from the ADAM family and which:

-   a) are a humanized variant of one of the amino acid sequences of SEQ     ID NO's: 868 to 973 and/or 1044 to 1053; and/or -   b) have at least 80% amino acid identity with at least one of the     amino acid sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053,     in which for the purposes of determining the degree of amino acid     identity, the amino acid residues that form the CDR sequences are     disregarded;     and in which: -   c) preferably one or more of the amino acid residues at positions     11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat     numbering are chosen from the Hallmark residues mentioned in Table     A-3 below.

According to another specific aspect of the invention, the invention provides a number of stretches of amino acid residues (i.e. small peptides) that are particularly suited for binding to a metalloproteinase from the ADAM family. These stretches of amino acid residues may be present in, and/or may be corporated into, an amino acid sequence of the invention, in particular in such a way that they form (part of) the antigen binding site of an amino acid sequence of the invention. As these stretches of amino acid residues were first generated as CDR sequences of heavy chain antibodies or V_(HH) sequences that were raised against a metalloproteinase from the ADAM family (or may be based on and/or derived from such CDR sequences, as further described herein), they will also generally be referred to herein as “CDR sequences” (i.e. as CDR1 sequences, CDR2 sequences and CDR3 sequences, respectively). It should however be noted that the invention in its broadest sense is not limited to a specific structural role or function that these stretches of amino acid residues may have in an amino acid sequence of the invention, as long as these stretches of amino acid residues allow the amino acid sequence of the invention to bind to a metalloproteinase from the ADAM family. Thus, generally, the invention in its broadest sense comprises any amino acid sequence that is capable of binding to a metalloproteinase from the ADAM family and that comprises one or more CDR sequences as described herein, and in particular a suitable combination of two or more such CDR sequences, that are suitably linked to each other via one or more further amino acid sequences, such that the entire amino acid sequence forms a binding domain and/or binding unit that is capable of binding to a metalloproteinase from the ADAM family. It should however also be noted that the presence of only one such CDR sequence in an amino acid sequence of the invention may by itself already be sufficient to provide an amino acid sequence of the invention that is capable of binding to a metalloproteinase from the ADAM family; reference is for example again made to the so-called “Expedite fragments” described in WO 03/050531.

Thus, in another specific, but non-limiting aspect, the amino acid sequence of the invention may be an amino acid sequence that comprises at least one amino acid sequence that is chosen from the group consisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences that are described herein (or any suitable combination thereof). In particular, an amino acid sequence of the invention may be an amino acid sequence that comprises at least one antigen binding site, wherein said antigen binding site comprises at least one amino acid sequence that is chosen from the group consisting of the CDR1 sequences, CDR2 sequences and CDR3 sequences that are described herein (or any suitable combination thereof).

Generally, in this aspect of the invention, the amino acid sequence of the invention may be any amino acid sequence that comprises at least one stretch of amino acid residues, in which said stretch of amino acid residues has an amino acid sequence that corresponds to the sequence of at least one of the CDR sequences described herein. Such an amino acid sequence may or may not comprise an immunoglobulin fold. For example, and without limitation, such an amino acid sequence may be a suitable fragment of an immunoglobulin sequence that comprises at least one such CDR sequence, but that is not large enough to form a (complete) immunoglobulin fold (reference is for example again made to the “Expedite fragments” described in WO 03/050531). Alternatively, such an amino acid sequence may be a suitable “protein scaffold” that comprises least one stretch of amino acid residues that corresponds to such a CDR sequence (i.e. as part of its antigen binding site). Suitable scaffolds for presenting amino acid sequences will be clear to the skilled person, and for example comprise, without limitation, to binding scaffolds based on or derived from immunoglobulins (i.e. other than the immunoglobulin sequences already described herein), protein scaffolds derived from protein A domains (such as Affibodies™), tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, designed ankyrin repeats, avimers and PDZ domains (Binz et al., Nat. Biotech 2005, Vol 23:1257), and binding moieties based on DNA or RNA including but not limited to DNA or RNA aptamers (Ulrich et al., Comb Chem High Throughput Screen 2006 9(8):619-32).

Again, any amino acid sequence of the invention that comprises one or more of these CDR sequences is preferably such that it can specifically bind (as defined herein) to a metalloproteinase from the ADAM family, and more in particular such that it can bind to a metalloproteinase from the ADAM family with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A) value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein), that is as defined herein.

More in particular, the amino acid sequences according to this aspect of the invention may be any amino acid sequence that comprises at least one antigen binding site, wherein said antigen binding site comprises at least two amino acid sequences that are chosen from the group consisting of the CDR1 sequences described herein, the CDR2 sequences described herein and the CDR3 sequences described herein, such that (i) when the first amino acid sequence is chosen from the CDR1 sequences described herein, the second amino acid sequence is chosen from the CDR2 sequences described herein or the CDR3 sequences described herein; (ii) when the first amino acid sequence is chosen from the CDR2 sequences described herein, the second amino acid sequence is chosen from the CDR1 sequences described herein or the CDR3 sequences described herein; or (iii) when the first amino acid sequence is chosen from the CDR3 sequences described herein, the second amino acid sequence is chosen from the CDR1 sequences described herein or the CDR3 sequences described herein.

Even more in particular, the amino acid sequences of the invention may be amino acid sequences that comprise at least one antigen binding site, wherein said antigen binding site comprises at least three amino acid sequences that are chosen from the group consisting of the CDR1 sequences described herein, the CDR2 sequences described herein and the CDR3 sequences described herein, such that the first amino acid sequence is chosen from the CDR1 sequences described herein, the second amino acid sequence is chosen from the CDR2 sequences described herein, and the third amino acid sequence is chosen from the CDR3 sequences described herein. Preferred combinations of CDR1, CDR2 and CDR3 sequences will become clear from the further description herein. As will be clear to the skilled person, such an amino acid sequence is preferably an immunoglobulin sequence (as further described herein), but it may for example also be any other amino acid sequence that comprises a suitable scaffold for presenting said CDR sequences.

Thus, in one specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against a metalloproteinase from the ADAM family, that comprises one or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     337; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     337; -   d) the amino acid sequences of SEQ ID NO's: 444 to 549; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     549; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     549; -   g) the amino acid sequences of SEQ ID NO's: 656 to 761; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     761; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     761;     or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more amino acid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence     according to b) and/or c) is preferably, and compared to the     corresponding amino acid sequence according to a), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to b) and/or c) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to a);     and/or -   iii) the amino acid sequence according to b) and/or c) may be an     amino acid sequence that is derived from an amino acid sequence     according to a) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence     according to e) and/or f) is preferably, and compared to the     corresponding amino acid sequence according to d), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to e) and/or f) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to d);     and/or -   iii) the amino acid sequence according to e) and/or f) may be an     amino acid sequence that is derived from an amino acid sequence     according to d) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence     according to h) and/or i) is preferably, and compared to the     corresponding amino acid sequence according to g), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to h) and/or i) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to g);     and/or -   iii) the amino acid sequence according to h) and/or i) may be an     amino acid sequence that is derived from an amino acid sequence     according to g) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs also generally apply to any amino acid sequences of the invention that comprise one or more amino acid sequences according to b), c), e), f), h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprises one or more stretches of amino acid residues chosen from the group consisting of:

-   i) the amino acid sequences of SEQ ID NO's: 232 to 337; -   ii) the amino acid sequences of SEQ ID NO's: 444 to 549; and -   iii) the amino acid sequences of SEQ ID NO's: 656 to 761;     or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of said stretches of amino acid residues forms part of the antigen binding site for binding against a metalloproteinase from the ADAM family.

In a more specific, but again non-limiting aspect, the invention relates to an amino acid sequence directed against a metalloproteinase from the ADAM family, that comprises two or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     337; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     337; -   d) the amino acid sequences of SEQ ID NO's: 444 to 549; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     549; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     549; -   g) the amino acid sequences of SEQ ID NO's: 656 to 761; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     761; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     761;     such that (i) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences according to a), b)     or c), the second stretch of amino acid residues corresponds to one     of the amino acid sequences according to d), e), f), g), h) or     i); (ii) when the first stretch of amino acid residues corresponds     to one of the amino acid sequences according to d), e) or f), the     second stretch of amino acid residues corresponds to one of the     amino acid sequences according to a), b), c), g), h) or i); or (iii)     when the first stretch of amino acid residues corresponds to one of     the amino acid sequences according to g), h) or i), the second     stretch of amino acid residues corresponds to one of the amino acid     sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprises two or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337; -   b) the amino acid sequences of SEQ ID NO's: 444 to 549; and -   c) the amino acid sequences of SEQ ID NO's: 656 to 761;     such that, (i) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 232     to 337, the second stretch of amino acid residues corresponds to one     of the amino acid sequences of SEQ ID NO's: 444 to 549 or of SEQ ID     NO's: 656 to 761; (ii) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 444     to 549, the second stretch of amino acid residues corresponds to one     of the amino acid sequences of SEQ ID NO's: 232 to 337 or of SEQ ID     NO's: 656 to 761; or (iii) when the first stretch of amino acid     residues corresponds to one of the amino acid sequences of SEQ ID     NO's: 656 to 761, the second stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 232     to 337 or of SEQ ID NO's: 444 to 549.

Also, in such an amino acid sequence, the at least two stretches of amino acid residues again preferably form part of the antigen binding site for binding against a metalloproteinase from the ADAM family.

In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against a metalloproteinase from the ADAM family, that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     337; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     337;     the second stretch of amino acid residues is chosen from the group     consisting of: -   d) the amino acid sequences of SEQ ID NO's: 444 to 549; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     549; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     549;     and the third stretch of amino acid residues is chosen from the     group consisting of: -   g) the amino acid sequences of SEQ ID NO's: 656 to 761; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     761; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     761.

Preferably, in this specific aspect, the first stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 232 to 337; the second stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 444 to 549; and the third stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 656 to 761.

Again, preferably, in such an amino acid sequence, the at least three stretches of amino acid residues forms part of the antigen binding site for binding against a metalloproteinase from the ADAM family.

Preferred combinations of such stretches of amino acid sequences will become clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053, in which the amino acid residues that form the framework regions are disregarded. Also, such amino acid sequences of the invention can be as further described herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to a metalloproteinase from the ADAM family; and more in particular bind to a metalloproteinase from the ADAM family with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     337;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     337;     and/or     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 549;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     549;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     549;     and/or     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 761;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     761;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     761.

In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 232 to 337; and/or CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 444 to 549; and/or CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 656 to 761.

In particular, when the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     337;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     337;     and     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 549;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     549;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     549;     and     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 761;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     761;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     761; or any suitable fragment of such an amino acid sequence

In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 232 to 337; and CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 444 to 549; and CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 656 to 761.

Again, preferred combinations of CDR sequences will become clear from the further description herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to a metalloproteinase from the ADAM family; and more in particular bind to a metalloproteinase from the ADAM family with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to an amino acid sequence that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053, in which the amino acid residues that form the framework regions are disregarded. Such amino acid sequences of the invention can be as further described herein.

In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against ADAM8 (such as a Nanobody of the invention, as further described herein), that comprises one or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     250; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     250; -   d) the amino acid sequences of SEQ ID NO's: 444 to 462; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     462; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     462; -   g) the amino acid sequences of SEQ ID NO's: 656 to 674; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     674; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     674;     or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more amino acid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence     according to b) and/or c) is preferably, and compared to the     corresponding amino acid sequence according to a), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to b) and/or c) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to a);     and/or -   iii) the amino acid sequence according to b) and/or c) may be an     amino acid sequence that is derived from an amino acid sequence     according to a) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence     according to e) and/or f) is preferably, and compared to the     corresponding amino acid sequence according to d), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to e) and/or f) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to d);     and/or -   iii) the amino acid sequence according to e) and/or f) may be an     amino acid sequence that is derived from an amino acid sequence     according to d) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence     according to h) and/or i) is preferably, and compared to the     corresponding amino acid sequence according to g), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to h) and/or i) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to g);     and/or -   iii) the amino acid sequence according to h) and/or i) may be an     amino acid sequence that is derived from an amino acid sequence     according to g) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs also generally apply to any amino acid sequences of the invention that comprise one or more amino acid sequences according to b), c), e), f), h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprises one or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250; -   b) the amino acid sequences of SEQ ID NO's: 444 to 462; and -   c) the amino acid sequences of SEQ ID NO's: 656 to 674;     or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of said stretches of amino acid residues forms part of the antigen binding site for binding against ADAM8.

In a more specific, but again non-limiting aspect, the invention relates to an amino acid sequence directed against ADAM8, that comprises two or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     250; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     250; -   d) the amino acid sequences of SEQ ID NO's: 444 to 462; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     462; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     462; -   g) the amino acid sequences of SEQ ID NO's: 656 to 674; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     674; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     674;     such that (i) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences according to a), b)     or c), the second stretch of amino acid residues corresponds to one     of the amino acid sequences according to d), e), f), g), h) or     i); (ii) when the first stretch of amino acid residues corresponds     to one of the amino acid sequences according to d), e) or f), the     second stretch of amino acid residues corresponds to one of the     amino acid sequences according to a), b), c), g), h) or i); or (iii)     when the first stretch of amino acid residues corresponds to one of     the amino acid sequences according to g), h) or i), the second     stretch of amino acid residues corresponds to one of the amino acid     sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprises two or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250; -   b) the amino acid sequences of SEQ ID NO's: 444 to 462; and -   c) the amino acid sequences of SEQ ID NO's: 656 to 674;     such that, (i) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 232     to 250, the second stretch of amino acid residues corresponds to one     of the amino acid sequences of SEQ ID NO's: 444 to 462 or of SEQ ID     NO's: 656 to 674; (ii) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 444     to 462, the second stretch of amino acid residues corresponds to one     of the amino acid sequences of SEQ ID NO's: 232 to 250 or of SEQ ID     NO's: 656 to 674; or (iii) when the first stretch of amino acid     residues corresponds to one of the amino acid sequences of SEQ ID     NO's: 656 to 674, the second stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 232     to 250 or of SEQ ID NO's: 444 to 462.

Also, in such an amino acid sequence, the at least two stretches of amino acid residues again preferably form part of the antigen binding site for binding against ADAM8.

In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against ADAM8, that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     250; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     250;     the second stretch of amino acid residues is chosen from the group     consisting of: -   d) the amino acid sequences of SEQ ID NO's: 444 to 462; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     462; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     462;     and the third stretch of amino acid residues is chosen from the     group consisting of: -   g) the amino acid sequences of SEQ ID NO's: 656 to 674; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     674; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     674.

Preferably, in this specific aspect, the first stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 232 to 250; the second stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 444 to 462; and the third stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 656 to 674.

Again, preferably, in such an amino acid sequence, the at least three stretches of amino acid residues forms part of the antigen binding site for binding against ADAM8.

Preferred combinations of such stretches of amino acid sequences will become clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 868 to 886. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 868 to 886, in which the amino acid residues that form the framework regions are disregarded. Also, such amino acid sequences of the invention can be as further described herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to ADAM8; and more in particular bind to ADAM8 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     250;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     250;     and/or     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 462;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     462;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     462;     and/or     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 674;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     674;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     674.

In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 232 to 250; and/or CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 444 to 462; and/or CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 656 to 674.

In particular, when the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     250;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     250;     and     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 462;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     462;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     462;     and     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 674;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     674;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     674; or any suitable fragment of such an amino acid sequence

In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 232 to 250; and CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 444 to 462; and CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 656 to 674.

Again, preferred combinations of CDR sequences will become clear from the further description herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to ADAM8; and more in particular bind to ADAM8 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to an amino acid sequence that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 868 to 886. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 868 to 886, in which the amino acid residues that form the framework regions are disregarded. Such amino acid sequences of the invention can be as further described herein.

In another more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against ADAM9 (such as a Nanobody of the invention, as further described herein), that comprises one or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to     987; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 251 to     271 and/or 984 to 987; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 251 to     271 and/or 984 to 987; -   d) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004     to 1007; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 463 to     483 and/or 1004 to 1007; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 463 to     483 and/or 1004 to 1007; -   g) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024     to 1027; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 675 to     695 and/or 1024 to 1027; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 675 to     695 and/or 1024 to 1027;     or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more amino acid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence     according to b) and/or c) is preferably, and compared to the     corresponding amino acid sequence according to a), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to b) and/or c) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to a);     and/or -   iii) the amino acid sequence according to b) and/or c) may be an     amino acid sequence that is derived from an amino acid sequence     according to a) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence     according to e) and/or f) is preferably, and compared to the     corresponding amino acid sequence according to d), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to e) and/or f) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to d);     and/or -   iii) the amino acid sequence according to e) and/or f) may be an     amino acid sequence that is derived from an amino acid sequence     according to d) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence     according to h) and/or i) is preferably, and compared to the     corresponding amino acid sequence according to g), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to h) and/or i) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to g);     and/or -   iii) the amino acid sequence according to h) and/or i) may be an     amino acid sequence that is derived from an amino acid sequence     according to g) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs also generally apply to any amino acid sequences of the invention that comprise one or more amino acid sequences according to b), c), e), f), h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprises one or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to     987; -   b) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004     to 1007; and -   c) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024     to 1027;     or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of said stretches of amino acid residues forms part of the antigen binding site for binding against ADAM9.

In a more specific, but again non-limiting aspect, the invention relates to an amino acid sequence directed against ADAM9, that comprises two or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to     987; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 251 to     271 and/or 984 to 987; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 251 to     271 and/or 984 to 987; -   d) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004     to 1007; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 463 to     483 and/or 1004 to 1007; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 463 to     483 and/or 1004 to 1007; -   g) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024     to 1027; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 675 to     695 and/or 1024 to 1027; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 675 to     695 and/or 1024 to 1027;     such that (i) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences according to a), b)     or c), the second stretch of amino acid residues corresponds to one     of the amino acid sequences according to d), e), f), g), h) or     i); (ii) when the first stretch of amino acid residues corresponds     to one of the amino acid sequences according to d), e) or f), the     second stretch of amino acid residues corresponds to one of the     amino acid sequences according to a), b), c), g), h) or i); or (iii)     when the first stretch of amino acid residues corresponds to one of     the amino acid sequences according to g), h) or i), the second     stretch of amino acid residues corresponds to one of the amino acid     sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprises two or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to     987; -   b) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004     to 1007; and -   c) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024     to 1027;     such that, (i) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 251     to 271 and/or 984 to 987, the second stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 463     to 483 and/or 1004 to 1007 or of SEQ ID NO's: 675 to 695 and/or 1024     to 1027; (ii) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 463     to 483 and/or 1004 to 1007, the second stretch of amino acid     residues corresponds to one of the amino acid sequences of SEQ ID     NO's: 251 to 271 and/or 984 to 987 or of SEQ ID NO's: 675 to 695     and/or 1024 to 1027; or (iii) when the first stretch of amino acid     residues corresponds to one of the amino acid sequences of SEQ ID     NO's: 675 to 695 and/or 1024 to 1027, the second stretch of amino     acid residues corresponds to one of the amino acid sequences of SEQ     ID NO's: 251 to 271 and/or 984 to 987 or of SEQ ID NO's: 463 to 483     and/or 1004 to 1007.

Also, in such an amino acid sequence, the at least two stretches of amino acid residues again preferably form part of the antigen binding site for binding against ADAM9.

In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against ADAM9, that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to     987; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 251 to     271 and/or 984 to 987; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 251 to     271 and/or 984 to 987;     the second stretch of amino acid residues is chosen from the group     consisting of: -   d) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004     to 1007; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 463 to     483 and/or 1004 to 1007; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 463 to     483 and/or 1004 to 1007;     and the third stretch of amino acid residues is chosen from the     group consisting of: -   g) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024     to 1027; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 675 to     695 and/or 1024 to 1027; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 675 to     695 and/or 1024 to 1027.

Preferably, in this specific aspect, the first stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to 987; the second stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004 to 1007; and the third stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024 to 1027.

Again, preferably, in such an amino acid sequence, the at least three stretches of amino acid residues forms part of the antigen binding site for binding against ADAM9.

Preferred combinations of such stretches of amino acid sequences will become clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 887 to 907 and 1044 to 1047. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 887 to 907 and 1044 to 1047, in which the amino acid residues that form the framework regions are disregarded. Also, such amino acid sequences of the invention can be as further described herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to ADAM9; and more in particular bind to ADAM9 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to     987;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 251 to     271 and/or 984 to 987;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 251 to     271 and/or 984 to 987;     and/or     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004     to 1007;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 463 to     483 and/or 1004 to 1007;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 463 to     483 and/or 1004 to 1007;     and/or     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024     to 1027;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 675 to     695 and/or 1024 to 1027;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 675 to     695 and/or 1024 to 1027.

In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to 987; and/or CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004 to 1007; and/or CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024 to 1027.

In particular, when the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to     987;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 251 to     271 and/or 984 to 987;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 251 to     271 and/or 984 to 987;     and     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004     to 1007;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 463 to     483 and/or 1004 to 1007;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 463 to     483 and/or 1004 to 1007;     and     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024     to 1027;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 675 to     695 and/or 1024 to 1027;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 675 to     695 and/or 1024 to 1027; or any suitable fragment of such an amino     acid sequence

In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to 987; and CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004 to 1007; and CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024 to 1027.

Again, preferred combinations of CDR sequences will become clear from the further description herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to ADAM9; and more in particular bind to ADAM9 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to an amino acid sequence that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 887 to 907 and 1044 to 1047. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 887 to 907 and 1044 to 1047, in which the amino acid residues that form the framework regions are disregarded. Such amino acid sequences of the invention can be as further described herein.

In another even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against ADAM10 (such as a Nanobody of the invention, as further described herein), that comprises one or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 272 to     295; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 272 to     295; -   d) the amino acid sequences of SEQ ID NO's: 484 to 507; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 484 to     507; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 484 to     507; -   g) the amino acid sequences of SEQ ID NO's: 696 to 719; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 696 to     719; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 696 to     719;     or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more amino acid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence     according to b) and/or c) is preferably, and compared to the     corresponding amino acid sequence according to a), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to b) and/or c) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to a);     and/or -   iii) the amino acid sequence according to b) and/or c) may be an     amino acid sequence that is derived from an amino acid sequence     according to a) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence     according to e) and/or f) is preferably, and compared to the     corresponding amino acid sequence according to d), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to e) and/or f) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to d);     and/or -   iii) the amino acid sequence according to e) and/or f) may be an     amino acid sequence that is derived from an amino acid sequence     according to d) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence     according to h) and/or i) is preferably, and compared to the     corresponding amino acid sequence according to g), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to h) and/or i) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to g);     and/or -   iii) the amino acid sequence according to h) and/or i) may be an     amino acid sequence that is derived from an amino acid sequence     according to g) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs also generally apply to any amino acid sequences of the invention that comprise one or more amino acid sequences according to b), c), e), f), h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprises one or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295; -   b) the amino acid sequences of SEQ ID NO's: 484 to 507; and -   c) the amino acid sequences of SEQ ID NO's: 696 to 719;     or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of said stretches of amino acid residues forms part of the antigen binding site for binding against ADAM10.

In a more specific, but again non-limiting aspect, the invention relates to an amino acid sequence directed against ADAM10, that comprises two or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 272 to     295; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 272 to     295; -   d) the amino acid sequences of SEQ ID NO's: 484 to 507; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 484 to     507; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 484 to     507; -   g) the amino acid sequences of SEQ ID NO's: 696 to 719; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 696 to     719; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 696 to     719;     such that (i) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences according to a), b)     or c), the second stretch of amino acid residues corresponds to one     of the amino acid sequences according to d), e), f), g), h) or     i); (ii) when the first stretch of amino acid residues corresponds     to one of the amino acid sequences according to d), e) or f), the     second stretch of amino acid residues corresponds to one of the     amino acid sequences according to a), b), c), g), h) or i); or (iii)     when the first stretch of amino acid residues corresponds to one of     the amino acid sequences according to g), h) or i), the second     stretch of amino acid residues corresponds to one of the amino acid     sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprises two or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295; -   b) the amino acid sequences of SEQ ID NO's: 484 to 507; and -   c) the amino acid sequences of SEQ ID NO's: 696 to 719;     such that, (i) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 272     to 295, the second stretch of amino acid residues corresponds to one     of the amino acid sequences of SEQ ID NO's: 484 to 507 or of SEQ ID     NO's: 696 to 719; (ii) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 484     to 507, the second stretch of amino acid residues corresponds to one     of the amino acid sequences of SEQ ID NO's: 272 to 295 or of SEQ ID     NO's: 696 to 719; or (iii) when the first stretch of amino acid     residues corresponds to one of the amino acid sequences of SEQ ID     NO's: 696 to 719, the second stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 272     to 295 or of SEQ ID NO's: 484 to 507.

Also, in such an amino acid sequence, the at least two stretches of amino acid residues again preferably form part of the antigen binding site for binding against ADAM10.

In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against ADAM10, that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 272 to     295; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 272 to     295;     the second stretch of amino acid residues is chosen from the group     consisting of: -   d) the amino acid sequences of SEQ ID NO's: 484 to 507; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 484 to     507; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 484 to     507;     and the third stretch of amino acid residues is chosen from the     group consisting of: -   g) the amino acid sequences of SEQ ID NO's: 696 to 719; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 696 to     719; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 696 to     719.

Preferably, in this specific aspect, the first stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 272 to 295; the second stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 484 to 507; and the third stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 696 to 719.

Again, preferably, in such an amino acid sequence, the at least three stretches of amino acid residues forms part of the antigen binding site for binding against ADAM10.

Preferred combinations of such stretches of amino acid sequences will become clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 908 to 931. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 908 to 931, in which the amino acid residues that form the framework regions are disregarded. Also, such amino acid sequences of the invention can be as further described herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to ADAM10; and more in particular bind to ADAM10 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 272 to     295;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 272 to     295;     and/or     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 484 to 507;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 484 to     507;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 484 to     507;     and/or     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 696 to 719;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 696 to     719;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 696 to     719.

In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 272 to 295; and/or CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 484 to 507; and/or CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 696 to 719.

In particular, when the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 272 to     295;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 272 to     295;     and     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 484 to 507;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 484 to     507;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 484 to     507;     and     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 696 to 719;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 696 to     719;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 696 to     719; or any suitable fragment of such an amino acid sequence

In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 272 to 295; and CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 484 to 507; and CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 696 to 719.

Again, preferred combinations of CDR sequences will become clear from the further description herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to ADAM10; and more in particular bind to ADAM10 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to an amino acid sequence that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 908 to 931. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 908 to 931, in which the amino acid residues that form the framework regions are disregarded. Such amino acid sequences of the invention can be as further described herein.

In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against ADAM17 (such as a Nanobody of the invention, as further described herein), that comprises one or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to     992; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 296 to     312 and/or 988 to 992; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 296 to     312 and/or 988 to 992; -   d) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008     to 1012; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 508 to     524 and/or 1008 to 1012; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 508 to     524 and/or 1008 to 1012; -   g) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028     to 1032; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 720 to     736 and/or 1028 to 1032; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 720 to     736 and/or 1028 to 1032;     or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more amino acid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence     according to b) and/or c) is preferably, and compared to the     corresponding amino acid sequence according to a), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to b) and/or c) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to a);     and/or -   iii) the amino acid sequence according to b) and/or c) may be an     amino acid sequence that is derived from an amino acid sequence     according to a) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence     according to e) and/or f) is preferably, and compared to the     corresponding amino acid sequence according to d), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to e) and/or f) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to d);     and/or -   iii) the amino acid sequence according to e) and/or f) may be an     amino acid sequence that is derived from an amino acid sequence     according to d) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence     according to h) and/or i) is preferably, and compared to the     corresponding amino acid sequence according to g), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to h) and/or i) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to g);     and/or -   iii) the amino acid sequence according to h) and/or i) may be an     amino acid sequence that is derived from an amino acid sequence     according to g) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs also generally apply to any amino acid sequences of the invention that comprise one or more amino acid sequences according to b), c), e), f), h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprises one or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to     992; -   b) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008     to 1012; and -   c) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028     to 1032;     or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of said stretches of amino acid residues forms part of the antigen binding site for binding against ADAM17.

In a more specific, but again non-limiting aspect, the invention relates to an amino acid sequence directed against ADAM17, that comprises two or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to     992; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 296 to     312 and/or 988 to 992; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 296 to     312 and/or 988 to 992; -   d) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008     to 1012; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 508 to     524 and/or 1008 to 1012; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 508 to     524 and/or 1008 to 1012; -   g) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028     to 1032; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 720 to     736 and/or 1028 to 1032; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 720 to     736 and/or 1028 to 1032;     such that (i) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences according to a), b)     or c), the second stretch of amino acid residues corresponds to one     of the amino acid sequences according to d), e), f), g), h) or     i); (ii) when the first stretch of amino acid residues corresponds     to one of the amino acid sequences according to d), e) or f), the     second stretch of amino acid residues corresponds to one of the     amino acid sequences according to a), b), c), g), h) or i); or (iii)     when the first stretch of amino acid residues corresponds to one of     the amino acid sequences according to g), h) or i), the second     stretch of amino acid residues corresponds to one of the amino acid     sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprises two or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to     992; -   b) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008     to 1012; and -   c) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028     to 1032;     such that, (i) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 296     to 312 and/or 988 to 992, the second stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 508     to 524 and/or 1008 to 1012 or of SEQ ID NO's: 720 to 736 and/or 1028     to 1032; (ii) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 508     to 524 and/or 1008 to 1012, the second stretch of amino acid     residues corresponds to one of the amino acid sequences of SEQ ID     NO's: 296 to 312 and/or 988 to 992 or of SEQ ID NO's: 720 to 736     and/or 1028 to 1032; or (iii) when the first stretch of amino acid     residues corresponds to one of the amino acid sequences of SEQ ID     NO's: 720 to 736 and/or 1028 to 1032, the second stretch of amino     acid residues corresponds to one of the amino acid sequences of SEQ     ID NO's: 296 to 312 and/or 988 to 992 or of SEQ ID NO's: 508 to 524     and/or 1008 to 1012.

Also, in such an amino acid sequence, the at least two stretches of amino acid residues again preferably form part of the antigen binding site for binding against ADAM17.

In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against ADAM17, that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to     992; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 296 to     312 and/or 988 to 992; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 296 to     312 and/or 988 to 992;     the second stretch of amino acid residues is chosen from the group     consisting of: -   d) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008     to 1012; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 508 to     524 and/or 1008 to 1012; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 508 to     524 and/or 1008 to 1012;     and the third stretch of amino acid residues is chosen from the     group consisting of: -   g) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028     to 1032; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 720 to     736 and/or 1028 to 1032; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 720 to     736 and/or 1028 to 1032.

Preferably, in this specific aspect, the first stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to 992; the second stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008 to 1012; and the third stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028 to 1032.

Again, preferably, in such an amino acid sequence, the at least three stretches of amino acid residues forms part of the antigen binding site for binding against ADAM17.

Preferred combinations of such stretches of amino acid sequences will become clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 932 to 948 and 1048 to 1052. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 932 to 948 and 1048 to 1052, in which the amino acid residues that form the framework regions are disregarded. Also, such amino acid sequences of the invention can be as further described herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to ADAM17; and more in particular bind to ADAM17 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC50 value, as further described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to     992;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 296 to     312 and/or 988 to 992;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 296 to     312 and/or 988 to 992;     and/or     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008     to 1012;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 508 to     524 and/or 1008 to 1012;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 508 to     524 and/or 1008 to 1012;     and/or     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028     to 1032;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 720 to     736 and/or 1028 to 1032;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 720 to     736 and/or 1028 to 1032.

In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to 992; and/or CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008 to 1012; and/or CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028 to 1032.

In particular, when the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to     992;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 296 to     312 and/or 988 to 992;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 296 to     312 and/or 988 to 992;     and     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008     to 1012;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 508 to     524 and/or 1008 to 1012;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 508 to     524 and/or 1008 to 1012;     and     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028     to 1032;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 720 to     736 and/or 1028 to 1032;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 720 to     736 and/or 1028 to 1032; or any suitable fragment of such an amino     acid sequence

In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to 992; and CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008 to 1012; and CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028 to 1032.

Again, preferred combinations of CDR sequences will become clear from the further description herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to ADAM17; and more in particular bind to ADAM17 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to an amino acid sequence that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 932 to 948 and 1048 to 1052. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 932 to 948 and 1048 to 1052, in which the amino acid residues that form the framework regions are disregarded. Such amino acid sequences of the invention can be as further described herein.

In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against ADAMTS5 (such as a Nanobody of the invention, as further described herein), that comprises one or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 or 993; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 313 to     337 or 993; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 313 to     337 or 993; -   d) the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 525 to     549 or 1013; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 525 to     549 or 1013; -   g) the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 737 to     761 or 1033; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 737 to     761 or 1033;     or any suitable combination thereof.

When an amino acid sequence of the invention contains one or more amino acid sequences according to b) and/or c):

-   i) any amino acid substitution in such an amino acid sequence     according to b) and/or c) is preferably, and compared to the     corresponding amino acid sequence according to a), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to b) and/or c) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to a);     and/or -   iii) the amino acid sequence according to b) and/or c) may be an     amino acid sequence that is derived from an amino acid sequence     according to a) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

Similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to e) and/or f):

-   i) any amino acid substitution in such an amino acid sequence     according to e) and/or f) is preferably, and compared to the     corresponding amino acid sequence according to d), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to e) and/or f) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to d);     and/or -   iii) the amino acid sequence according to e) and/or f) may be an     amino acid sequence that is derived from an amino acid sequence     according to d) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

Also, similarly, when an amino acid sequence of the invention contains one or more amino acid sequences according to h) and/or i):

-   i) any amino acid substitution in such an amino acid sequence     according to h) and/or i) is preferably, and compared to the     corresponding amino acid sequence according to g), a conservative     amino acid substitution, (as defined herein);     and/or -   ii) the amino acid sequence according to h) and/or i) preferably     only contains amino acid substitutions, and no amino acid deletions     or insertions, compared to the corresponding amino acid sequence     according to g);     and/or -   iii) the amino acid sequence according to h) and/or i) may be an     amino acid sequence that is derived from an amino acid sequence     according to g) by means of affinity maturation using one or more     techniques of affinity maturation known per se.

It should be understood that the last preceding paragraphs also generally apply to any amino acid sequences of the invention that comprise one or more amino acid sequences according to b), c), e), f), h) or i), respectively.

In this specific aspect, the amino acid sequence preferably comprises one or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 or 993; -   b) the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013; and -   c) the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033;     or any suitable combination thereof.

Also, preferably, in such an amino acid sequence, at least one of said stretches of amino acid residues forms part of the antigen binding site for binding against ADAMTS5.

In a more specific, but again non-limiting aspect, the invention relates to an amino acid sequence directed against ADAMTS5, that comprises two or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 or 993; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 313 to     337 or 993; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 313 to     337 or 993; -   d) the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 525 to     549 or 1013; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 525 to     549 or 1013; -   g) the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 737 to     761 or 1033; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 737 to     761 or 1033;     such that (i) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences according to a), b)     or c), the second stretch of amino acid residues corresponds to one     of the amino acid sequences according to d), e), f), g), h) or     i); (ii) when the first stretch of amino acid residues corresponds     to one of the amino acid sequences according to d), e) or f), the     second stretch of amino acid residues corresponds to one of the     amino acid sequences according to a), b), c), g), h) or i); or (iii)     when the first stretch of amino acid residues corresponds to one of     the amino acid sequences according to g), h) or i), the second     stretch of amino acid residues corresponds to one of the amino acid     sequences according to a), b), c), d), e) or f).

In this specific aspect, the amino acid sequence preferably comprises two or more stretches of amino acid residues chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 or 993; -   b) the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013; and -   c) the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033;     such that, (i) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 313     to 337 or 993, the second stretch of amino acid residues corresponds     to one of the amino acid sequences of SEQ ID NO's: 525 to 549 or     1013 or of SEQ ID NO's: 737 to 761 or 1033; (ii) when the first     stretch of amino acid residues corresponds to one of the amino acid     sequences of SEQ ID NO's: 525 to 549 or 1013, the second stretch of     amino acid residues corresponds to one of the amino acid sequences     of SEQ ID NO's: 313 to 337 or 993 or of SEQ ID NO's: 737 to 761 or     1033; or (iii) when the first stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 737     to 761 or 1033, the second stretch of amino acid residues     corresponds to one of the amino acid sequences of SEQ ID NO's: 313     to 337 or 993 or of SEQ ID NO's: 525 to 549 or 1013.

Also, in such an amino acid sequence, the at least two stretches of amino acid residues again preferably form part of the antigen binding site for binding against ADAMTS5.

In an even more specific, but non-limiting aspect, the invention relates to an amino acid sequence directed against ADAMTS5, that comprises three or more stretches of amino acid residues, in which the first stretch of amino acid residues is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 or 993; -   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 313 to     337 or 993; -   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 313 to     337 or 993;     the second stretch of amino acid residues is chosen from the group     consisting of: -   d) the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013; -   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 525 to     549 or 1013; -   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 525 to     549 or 1013;     and the third stretch of amino acid residues is chosen from the     group consisting of: -   g) the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033; -   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 737 to     761 or 1033; -   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 737 to     761 or 1033.

Preferably, in this specific aspect, the first stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 313 to 337 or 993; the second stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013; and the third stretch of amino acid residues is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033.

Again, preferably, in such an amino acid sequence, the at least three stretches of amino acid residues forms part of the antigen binding site for binding against ADAMTS5.

Preferred combinations of such stretches of amino acid sequences will become clear from the further disclosure herein.

Preferably, in such amino acid sequences the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 949 to 974 and 1053. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 949 to 974 and 1053, in which the amino acid residues that form the framework regions are disregarded. Also, such amino acid sequences of the invention can be as further described herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to ADAMTS5; and more in particular bind to ADAMTS5 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

When the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 or 993;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 313 to     337 or 993;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 313 to     337 or 993;     and/or     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 525 to     549 or 1013;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 525 to     549 or 1013;     and/or     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 737 to     761 or 1033;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 737 to     761 or 1033.

In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 313 to 337 or 993; and/or CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013; and/or CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033.

In particular, when the amino acid sequence of the invention essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), the amino acid sequence of the invention is preferably such that:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 or 993;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 313 to     337 or 993;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 313 to     337 or 993;     and     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 525 to     549 or 1013;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 525 to     549 or 1013;     and     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 737 to     761 or 1033;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 737 to     761 or 1033; or any suitable fragment of such an amino acid sequence

In particular, such an amino acid sequence of the invention may be such that CDR1 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 313 to 337 or 993; and CDR2 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 525 to 549 or 1013; and CDR3 is chosen from the group consisting of the amino acid sequences of SEQ ID NO's: 737 to 761 or 1033.

Again, preferred combinations of CDR sequences will become clear from the further description herein.

Also, such amino acid sequences are preferably such that they can specifically bind (as defined herein) to ADAMTS5; and more in particular bind to ADAMTS5 with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

In one preferred, but non-limiting aspect, the invention relates to an amino acid sequence that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which the CDR sequences of said amino acid sequence have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 949 to 974 and 1053. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said amino acid sequence and one or more of the sequences of SEQ ID NO's: 949 to 974 and 1053, in which the amino acid residues that form the framework regions are disregarded. Such amino acid sequences of the invention can be as further described herein.

In such an amino acid sequence of the invention, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.

The framework sequences are preferably (a suitable combination of) immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by humanization or camelization). For example, the framework sequences may be framework sequences derived from a light chain variable domain (e.g. a V_(L)-sequence) and/or from a heavy chain variable domain (e.g. a V_(H)-sequence). In one particularly preferred aspect, the framework sequences are either framework sequences that have been derived from a V_(HH)-sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional V_(H) sequences that have been camelized (as defined herein).

The framework sequences are preferably such that the amino acid sequence of the invention is a domain antibody (or an amino acid sequence that is suitable for use as a domain antibody); is a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody); is a “dAb” (or an amino acid sequence that is suitable for use as a dAb); or is a Nanobody™ (including but not limited to V_(HH) sequence). Again, suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.

In particular, the framework sequences present in the amino acid sequences of the invention may contain one or more of Hallmark residues (as defined herein), such that the amino acid sequence of the invention is a Nanobody™. Some preferred, but non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.

Again, as generally described herein for the amino acid sequences of the invention, it is also possible to use suitable fragments (or combinations of fragments) of any of the foregoing, such as fragments that contain one or more CDR sequences, suitably flanked by and/or linked via one or more framework sequences (for example, in the same order as these CDR's and framework sequences may occur in the full-sized immunoglobulin sequence from which the fragment has been derived). Such fragments may also again be such that they comprise or can form an immunoglobulin fold, or alternatively be such that they do not comprise or cannot form an immunoglobulin fold.

In one specific aspect, such a fragment comprises a single CDR sequence as described herein (and in particular a CDR3 sequence), that is flanked on each side by (part of) a framework sequence (and in particular, part of the framework sequence(s) that, in the immunoglobulin sequence from which the fragment is derived, are adjacent to said CDR sequence. For example, a CDR3 sequence may be preceded by (part of) a FR3 sequence and followed by (part of) a FR4 sequence). Such a fragment may also contain a disulphide bridge, and in particular a disulphide bridge that links the two framework regions that precede and follow the CDR sequence, respectively (for the purpose of forming such a disulphide bridge, cysteine residues that naturally occur in said framework regions may be used, or alternatively cysteine residues may be synthetically added to or introduced into said framework regions). For a further description of these “Expedite fragments”, reference is again made to WO 03/050531, as well as to as well as to the U.S. provisional application of Ablynx N.V. entitled “Peptides capable of binding to serum proteins” of Ablynx N.V. (inventors: Revets, Hilde Adi Pierrette; Kolkman, Joost Alexander; and Hoogenboom, Hendricus Renerus Jacobus Mattheus) filed on Dec. 5, 2006 (see also PCT/EP2007/063348).

In another aspect, the invention relates to a compound or construct, and in particular a protein or polypeptide (also referred to herein as a “compound of the invention” or “polypeptide of the invention”, respectively) that comprises or essentially consists of one or more amino acid sequences of the invention (or suitable fragments thereof), and optionally further comprises one or more other groups, residues, moieties or binding units. As will become clear to the skilled person from the further disclosure herein, such further groups, residues, moieties, binding units or amino acid sequences may or may not provide further functionality to the amino acid sequence of the invention (and/or to the compound or construct in which it is present) and may or may not modify the properties of the amino acid sequence of the invention.

For example, such further groups, residues, moieties or binding units may be one or more additional amino acid sequences, such that the compound or construct is a (fusion) protein or (fusion) polypeptide. In a preferred but non-limiting aspect, said one or more other groups, residues, moieties or binding units are immunoglobulin sequences. Even more preferably, said one or more other groups, residues, moieties or binding units are chosen from the group consisting of domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, “dAb's”, amino acid sequences that are suitable for use as a dAb, or Nanobodies.

Alternatively, such groups, residues, moieties or binding units may for example be chemical groups, residues, moieties, which may or may not by themselves be biologically and/or pharmacologically active. For example, and without limitation, such groups may be linked to the one or more amino acid sequences of the invention so as to provide a “derivative” of an amino acid sequence or polypeptide of the invention, as further described herein.

Also within the scope of the present invention are compounds or constructs, that comprises or essentially consists of one or more derivatives as described herein, and optionally further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more linkers. Preferably, said one or more other groups, residues, moieties or binding units are amino acid sequences.

In the compounds or constructs described above, the one or more amino acid sequences of the invention and the one or more groups, residues, moieties or binding units may be linked directly to each other and/or via one or more suitable linkers or spacers. For example, when the one or more groups, residues, moieties or binding units are amino acid sequences, the linkers may also be amino acid sequences, so that the resulting compound or construct is a fusion (protein) or fusion (polypeptide).

As will be clear from the further description above and herein, this means that the amino acid sequences of the invention can be used as “building blocks” to form polypeptides of the invention, i.e. by suitably combining them with other groups, residues, moieties or binding units, in order to form compounds or constructs as described herein (such as, without limitations, the biparatopic. bi/multivalent and bi/multispecific polypeptides of the invention described herein) which combine within one molecule one or more desired properties or biological functions.

The compounds or polypeptides of the invention can generally be prepared by a method which comprises at least one step of suitably linking the one or more amino acid sequences of the invention to the one or more further groups, residues, moieties or binding units, optionally via the one or more suitable linkers, so as to provide the compound or polypeptide of the invention. Polypeptides of the invention can also be prepared by a method which generally comprises at least the steps of providing a nucleic acid that encodes a polypeptide of the invention, expressing said nucleic acid in a suitable manner, and recovering the expressed polypeptide of the invention. Such methods can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the methods and techniques further described herein.

The process of designing/selecting and/or preparing a compound or polypeptide of the invention, starting from an amino acid sequence of the invention, is also referred to herein as “formatting” said amino acid sequence of the invention; and an amino acid of the invention that is made part of a compound or polypeptide of the invention is said to be “formatted” or to be “in the format of” said compound or polypeptide of the invention. Examples of ways in which an amino acid sequence of the invention can be formatted and examples of such formats will be clear to the skilled person based on the disclosure herein; and such formatted amino acid sequences form a further aspect of the invention.

For example, a multispecific polypeptide of the invention (as defined herein) may at least comprise at least one amino acid sequence (such as a Nanobody) against an ADAM proteinase and at least one binding unit against TNF (such as the Nanobodies described in the international applications WO 06/122786 and WO 04/041862 of Ablynx N.V. or the dAb's described in WO 03/002609 or WO 04/003019). Such a multispecific polypeptide may for example be used in the prevention and treatment of inflammation and other diseases and disorders associated with TNF and the TNF pathways. Reference is made to WO 06/122786, WO 04/041862, WO 03/002609 or WO 04/003019 and WO 06/038027.

Another multispecific polypeptide of the invention may at least comprise at least one amino acid sequence (such as a Nanobody) against an ADAM proteinase (which amino acid sequence is preferably not inactivating) and at least one binding unit against a receptor so as to induce shedding and inactivation of said receptor. For example, such a multispecific polypeptide of the invention (as defined herein) may at least comprise at least one amino acid sequence (such as a Nanobody) against an ADAM proteinase (which amino acid sequence is preferably not inactivating) and at least one binding unit against a TNF receptor (e.g. TNFR1 or TNFR2) so as to induce shedding and inactivation of said TNF receptor (such as the dAb's described in WO 06/038027). Such a multispecific polypeptide may for example be used in the prevention and treatment of inflammation and other diseases and disorders associated with TNF and the TNF pathways. Reference is again made to WO 06/122786, WO 04/041862, WO 03/002609 or WO 04/003019 and WO 06/038027.

Another multispecific polypeptide of the invention may at least comprise at least one amino acid sequence (such as a Nanobody) against an ADAM proteinase (which amino acid sequence is preferably not inactivating) and at least one binding unit against APP so as to cleave or solubilize APP aggregates.

Another multispecific polypeptide of the invention may at least comprise at least two amino acid sequence (such as two Nanobodies) against different ADAM proteinase so as to provide an increased and potentially synergistic modulating (and preferably inhibiting) effect on the ADAM proteinases.

In one specific aspect of the invention, a compound of the invention or a polypeptide of the invention may have an increased half-life, compared to the corresponding amino acid sequence of the invention. Some preferred, but non-limiting examples of such compounds and polypeptides will become clear to the skilled person based on the further disclosure herein, and for example comprise amino acid sequences or polypeptides of the invention that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); amino acid sequences of the invention that comprise at least one additional binding site for binding to a serum protein (such as serum albumin); or polypeptides of the invention that comprise at least one amino acid sequence of the invention that is linked to at least one moiety (and in particular at least one amino acid sequence) that increases the half-life of the amino acid sequence of the invention. Examples of polypeptides of the invention that comprise such half-life extending moieties or amino acid sequences will become clear to the skilled person based on the further disclosure herein; and for example include, without limitation, polypeptides in which the one or more amino acid sequences of the invention are suitable linked to one or more serum proteins or fragments thereof (such as (human) serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, domain antibodies, amino acid sequences that are suitable for use as a domain antibody, single domain antibodies, amino acid sequences that are suitable for use as a single domain antibody, “dAb's”, amino acid sequences that are suitable for use as a dAb, or Nanobodies that can bind to serum proteins such as serum albumin (such as human serum albumin), serum immunoglobulins such as IgG, or transferrin; reference is made to the further description and references mentioned herein); polypeptides in which an amino acid sequence of the invention is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more amino acid sequences of the invention are suitable linked to one or more small proteins or peptides that can bind to serum proteins (such as, without limitation, the proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489 and to the U.S. provisional application of Ablynx N.V. entitled “Peptides capable of binding to serum proteins” of Ablynx N.V. filed on Dec. 5, 2006 (see also PCT/EP2007/063348).

Generally, the compounds or polypeptides of the invention with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence of the invention per se. For example, the compounds or polypeptides of the invention with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such compounds or polypeptides of the invention have a serum half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.

In another preferred, but non-limiting aspect of the invention, such compounds or polypeptides of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

In another aspect, the invention relates to a nucleic acid that encodes an amino acid sequence of the invention or a polypeptide of the invention (or a suitable fragment thereof). Such a nucleic acid will also be referred to herein as a “nucleic acid of the invention” and may for example be in the form of a genetic construct, as further described herein.

In another aspect, the invention relates to a host or host cell that expresses (or that under suitable circumstances is capable of expressing) an amino acid sequence of the invention and/or a polypeptide of the invention; and/or that contains a nucleic acid of the invention. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein.

The invention further relates to a product or composition containing or comprising at least one amino acid sequence of the invention, at least one polypeptide of the invention (or a suitable fragment thereof) and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se, i.e. depending on the intended use of the composition. Such a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein.

The invention further relates to methods for preparing or generating the amino acid sequences, polypeptides, nucleic acids, host cells, products and compositions described herein. Some preferred but non-limiting examples of such methods will become clear from the further description herein.

Generally, these methods may comprise the steps of:

-   a) providing a set, collection or library of amino acid sequences;     and -   b) screening said set, collection or library of amino acid sequences     for amino acid sequences that can bind to and/or have affinity for a     metalloproteinase from the ADAM family;     and -   c) isolating the amino acid sequence(s) that can bind to and/or have     affinity for a metalloproteinase from the ADAM family.

In such a method, the set, collection or library of amino acid sequences may be any suitable set, collection or library of amino acid sequences. For example, the set, collection or library of amino acid sequences may be a set, collection or library of immunoglobulin sequences (as described herein), such as a naïve set, collection or library of immunoglobulin sequences; a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

Also, in such a method, the set, collection or library of amino acid sequences may be a set, collection or library of heavy chain variable domains (such as V_(H) domains or V_(HH) domains) or of light chain variable domains. For example, the set, collection or library of amino acid sequences may be a set, collection or library of domain antibodies or single domain antibodies, or may be a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of amino acid sequences may be an immune set, collection or library of immunoglobulin sequences, for example derived from a mammal that has been suitably immunized with a metalloproteinase from the ADAM family or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of amino acid sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating amino acid sequences comprises at least the steps of:

-   a) providing a collection or sample of cells expressing amino acid     sequences; -   b) screening said collection or sample of cells for cells that     express an amino acid sequence that can bind to and/or have affinity     for a metalloproteinase from the ADAM family;     and -   c) either (i) isolating said amino acid sequence; or (ii) isolating     from said cell a nucleic acid sequence that encodes said amino acid     sequence, followed by expressing said amino acid sequence.

For example, when the desired amino acid sequence is an immunoglobulin sequence, the collection or sample of cells may for example be a collection or sample of B-cells. Also, in this method, the sample of cells may be derived from a mammal that has been suitably immunized with a metalloproteinase from the ADAM family or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The above method may be performed in any suitable manner, as will be clear to the skilled person. Reference is for example made to EP 0 542 810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of step b) is preferably performed using a flow cytometry technique such as FACS. For this, reference is for example made to Lieby et al., Blood, Vol. 97, No. 12, 3820 (2001).

In another aspect, the method for generating an amino acid sequence directed against a metalloproteinase from the ADAM family may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences     encoding amino acid sequences; -   b) screening said set, collection or library of nucleic acid     sequences for nucleic acid sequences that encode an amino acid     sequence that can bind to and/or has affinity for a     metalloproteinase from the ADAM family;     and -   c) isolating said nucleic acid sequence, followed by expressing said     amino acid sequence.

In such a method, the set, collection or library of nucleic acid sequences encoding amino acid sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of immunoglobulin sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of immunoglobulin sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of immunoglobulin sequences that have been subjected to affinity maturation.

Also, in such a method, the set, collection or library of nucleic acid sequences may encode a set, collection or library of heavy chain variable domains (such as V_(H) domains or V_(HH) domains) or of light chain variable domains. For example, the set, collection or library of nucleic acid sequences may encode a set, collection or library of domain antibodies or single domain antibodies, or a set, collection or library of amino acid sequences that are capable of functioning as a domain antibody or single domain antibody.

In a preferred aspect of this method, the set, collection or library of amino acid sequences may be an immune set, collection or library of nucleic acid sequences, for example derived from a mammal that has been suitably immunized with a metalloproteinase from the ADAM family or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The set, collection or library of nucleic acid sequences may for example encode an immune set, collection or library of heavy chain variable domains or of light chain variable domains. In one specific aspect, the set, collection or library of nucleotide sequences may encode a set, collection or library of V_(HH) sequences.

In the above methods, the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

The invention also relates to amino acid sequences that are obtained by the above methods, or alternatively by a method that comprises the one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said immunoglobulin sequence; and of expressing or synthesizing said amino acid sequence in a manner known per se, such as by expression in a suitable host cell or host organism or by chemical synthesis.

Also, following the steps above, one or more amino acid sequences of the invention may be suitably humanized (or alternatively camelized); and/or the amino acid sequence(s) thus obtained may be linked to each other or to one or more other suitable amino acid sequences (optionally via one or more suitable linkers) so as to provide a polypeptide of the invention. Also, a nucleic acid sequence encoding an amino acid sequence of the invention may be suitably humanized (or alternatively camelized) and suitably expressed; and/or one or more nucleic acid sequences encoding an amino acid sequence of the invention may be linked to each other or to one or more nucleic acid sequences that encode other suitable amino acid sequences (optionally via nucleotide sequences that encode one or more suitable linkers), after which the nucleotide sequence thus obtained may be suitably expressed so as to provide a polypeptide of the invention.

The invention further relates to applications and uses of the amino acid sequences, polypeptides, nucleic acids, host cells, products and compositions described herein, as well as to methods for the prevention and/or treatment for diseases and disorders associated with a metalloproteinase from the ADAM family. Some preferred but non-limiting applications and uses will become clear from the further description herein.

Other aspects, embodiments, advantages and applications of the invention will also become clear from the further description herein, in which the invention will be described and discussed in more detail with reference to the Nanobodies of the invention and polypeptides of the invention comprising the same, which form some of the preferred aspects of the invention.

As will become clear from the further description herein, Nanobodies generally offer certain advantages (outlined herein) compared to “dAb's” or similar (single) domain antibodies or immunoglobulin sequences, which advantages are also provided by the Nanobodies of the invention. However, it will be clear to the skilled person that the more general aspects of the teaching below can also be applied (either directly or analogously) to other amino acid sequences of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In the present description, examples and claims:

-   a) Unless indicated or defined otherwise, all terms used have their     usual meaning in the art, which will be clear to the skilled person.     Reference is for example made to the standard handbooks, such as     Sambrook et al, “Molecular Cloning: A Laboratory Manual” (2nd.Ed.),     Vols. 1-3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et     al, eds., “Current protocols in molecular biology”, Green Publishing     and Wiley Interscience, New York (1987); Lewin, “Genes II”, John     Wiley & Sons, New York, N.Y., (1985); Old et al., “Principles of     Gene Manipulation: An Introduction to Genetic Engineering”, 2nd     edition, University of California Press, Berkeley, Calif. (1981);     Roitt et al., “Immunology” (6th. Ed.), Mosby/Elsevier, Edinburgh     (2001); Roitt et al., Roitt's Essential Immunology, 10^(th) Ed.     Blackwell Publishing, UK (2001); and Janeway et al., “Immunobiology”     (6th Ed.), Garland Science Publishing/Churchill Livingstone, N.Y.     (2005), as well as to the general background art cited herein; -   b) Unless indicated otherwise, the term “immunoglobulin     sequence”—whether used herein to refer to a heavy chain antibody or     to a conventional 4-chain antibody—is used as a general term to     include both the full-size antibody, the individual chains thereof,     as well as all parts, domains or fragments thereof (including but     not limited to antigen-binding domains or fragments such as V_(HH)     domains or V_(H)/V_(L) domains, respectively). In addition, the term     “sequence” as used herein (for example in terms like “immunoglobulin     sequence”, “antibody sequence”, “variable domain sequence”, “V_(HH)     sequence” or “protein sequence”), should generally be understood to     include both the relevant amino acid sequence as well as nucleic     acids or nucleotide sequences encoding the same, unless the context     requires a more limited interpretation. Also, the term “nucleotide     sequence” as used herein also encompasses a nucleic acid molecule     with said nucleotide sequence, so that the terms “nucleotide     sequence” and “nucleic acid” should be considered equivalent and are     used interchangeably herein; -   c) Unless indicated otherwise, all methods, steps, techniques and     manipulations that are not specifically described in detail can be     performed and have been performed in a manner known per se, as will     be clear to the skilled person. Reference is for example again made     to the standard handbooks and the general background art mentioned     herein and to the further references cited therein; as well as to     for example the following reviews Presta, Adv. Drug Deliv. Rev.     2006, 58 (5-6): 640-56; Levin and Weiss, Mol. Biosyst. 2006, 2(1):     49-57; Irving et al., J. Immunol. Methods, 2001, 248(1-2), 31-45;     Schmitz et al., Placenta, 2000, 21 Suppl. A, S106-12, Gonzales et     al., Tumour Biol., 2005, 26(1), 31-43, which describe techniques for     protein engineering, such as affinity maturation and other     techniques for improving the specificity and other desired     properties of proteins such as immunoglobulins. -   d) Amino acid residues will be indicated according to the standard     three-letter or one-letter amino acid code, as mentioned in Table     A-2;

TABLE A-2 one-letter and three-letter amino acid code Nonpolar, Alanine Ala A uncharged Valine Val V (at pH 6.0-7.0)⁽³⁾ Leucine Leu L Isoleucine Ile I Phenylalanine Phe F Methionine⁽¹⁾ Met M Tryptophan Trp W Proline Pro P Polar, Glycine⁽²⁾ Gly G uncharged Serine Ser S (at pH 6.0-7.0) Threonine Thr T Cysteine Cys C Asparagine Asn N Glutamine Gln Q Tyrosine Tyr Y Polar, Lysine Lys K charged Arginine Arg R (at pH 6.0-7.0) Histidine⁽⁴⁾ His H Aspartate Asp D Glutamate Glu E Notes: ⁽¹⁾Sometimes also considered to be a polar uncharged amino acid. ⁽²⁾Sometimes also considered to be a nonpolar uncharged amino acid. ⁽³⁾As will be clear to the skilled person, the fact that an amino acid residue is referred to in this Table as being either charged or uncharged at pH 6.0 to 7.0 does not reflect in any way on the charge said amino acid residue may have at a pH lower than 6.0 and/or at a pH higher than 7.0; the amino acid residues mentioned in the Table can be either charged and/or uncharged at such a higher or lower pH, as will be clear to the skilled person. ⁽⁴⁾As is known in the art, the charge of a His residue is greatly dependant upon even small shifts in pH, but a His residue can generally be considered essentially uncharged at a pH of about 6.5.

-   e) For the purposes of comparing two or more nucleotide sequences,     the percentage of “sequence identity” between a first nucleotide     sequence and a second nucleotide sequence may be calculated by     dividing [the number of nucleotides in the first nucleotide sequence     that are identical to the nucleotides at the corresponding positions     in the second nucleotide sequence] by [the total number of     nucleotides in the first nucleotide sequence] and multiplying by     [100%], in which each deletion, insertion, substitution or addition     of a nucleotide in the second nucleotide sequence—compared to the     first nucleotide sequence—is considered as a difference at a single     nucleotide (position).     -   Alternatively, the degree of sequence identity between two or         more nucleotide sequences may be calculated using a known         computer algorithm for sequence alignment such as NCBI Blast         v2.0, using standard settings.     -   Some other techniques, computer algorithms and settings for         determining the degree of sequence identity are for example         described in WO 04/037999, EP 0 967 284, EP 1 085 089, WO         00/55318, WO 00/78972, WO 98/49185 and GB 2 357 768-A.     -   Usually, for the purpose of determining the percentage of         “sequence identity” between two nucleotide sequences in         accordance with the calculation method outlined hereinabove, the         nucleotide sequence with the greatest number of nucleotides will         be taken as the “first” nucleotide sequence, and the other         nucleotide sequence will be taken as the “second” nucleotide         sequence; -   f) For the purposes of comparing two or more amino acid sequences,     the percentage of “sequence identity” between a first amino acid     sequence and a second amino acid sequence (also referred to herein     as “amino acid identity”) may be calculated by dividing [the number     of amino acid residues in the first amino acid sequence that are     identical to the amino acid residues at the corresponding positions     in the second amino acid sequence] by [the total number of amino     acid residues in the first amino acid sequence] and multiplying by     [100%], in which each deletion, insertion, substitution or addition     of an amino acid residue in the second amino acid sequence—compared     to the first amino acid sequence—is considered as a difference at a     single amino acid residue (position), i.e. as an “amino acid     difference” as defined herein.     -   Alternatively, the degree of sequence identity between two amino         acid sequences may be calculated using a known computer         algorithm, such as those mentioned above for determining the         degree of sequence identity for nucleotide sequences, again         using standard settings.     -   Usually, for the purpose of determining the percentage of         “sequence identity” between two amino acid sequences in         accordance with the calculation method outlined hereinabove, the         amino acid sequence with the greatest number of amino acid         residues will be taken as the “first” amino acid sequence, and         the other amino acid sequence will be taken as the “second”         amino acid sequence.     -   Also, in determining the degree of sequence identity between two         amino acid sequences, the skilled person may take into account         so-called “conservative” amino acid substitutions, which can         generally be described as amino acid substitutions in which an         amino acid residue is replaced with another amino acid residue         of similar chemical structure and which has little or         essentially no influence on the function, activity or other         biological properties of the polypeptide. Such conservative         amino acid substitutions are well known in the art, for example         from WO 04/037999, GB-A-3 357 768, WO 98/49185, WO 00/46383 and         WO 01/09300; and (preferred) types and/or combinations of such         substitutions may be selected on the basis of the pertinent         teachings from WO 04/037999 as well as WO 98/49185 and from the         further references cited therein.     -   Such conservative substitutions preferably are substitutions in         which one amino acid within the following groups (a)-(e) is         substituted by another amino acid residue within the same         group: (a) small aliphatic, nonpolar or slightly polar residues:         Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged         residues and their (uncharged) amides: Asp, Asn, Glu and         Gln; (c) polar, positively charged residues: His, Arg and         Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val         and Cys; and (e) aromatic residues: Phe, Tyr and Trp.     -   Particularly preferred conservative substitutions are as         follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gln or         into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into         Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile         into Leu or into Val; Leu into Ile or into Val; Lys into Arg,         into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe         into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp         into Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into         Leu.     -   Any amino acid substitutions applied to the polypeptides         described herein may also be based on the analysis of the         frequencies of amino acid variations between homologous proteins         of different species developed by Schulz et al., Principles of         Protein Structure, Springer-Verlag, 1978, on the analyses of         structure forming potentials developed by Chou and Fasman,         Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978,         and on the analysis of hydrophobicity patterns in proteins         developed by Eisenberg et al., Proc. Natl. Acad. Sci. USA 81:         140-144, 1984; Kyte & Doolittle; J Molec. Biol. 157: 105-132,         198 1, and Goldman et al., Ann Rev. Biophys. Chem. 15: 321-353,         1986, all incorporated herein in their entirety by reference.         Information on the primary, secondary and tertiary structure of         Nanobodies is given in the description herein and in the general         background art cited above. Also, for this purpose, the crystal         structure of a V_(HH) domain from a llama is for example given         by Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803         (1996); Spinelli et al., Natural Structural Biology (1996); 3,         752-757; and Decanniere et al., Structure, Vol. 7, 4, 361         (1999). Further information about some of the amino acid         residues that in conventional V_(H) domains form the V_(H)/V_(L)         interface and potential camelizing substitutions on these         positions can be found in the prior art cited above. -   g) Amino acid sequences and nucleic acid sequences are said to be     “exactly the same” if they have 100% sequence identity (as defined     herein) over their entire length; -   h) When comparing two amino acid sequences, the term “amino acid     difference” refers to an insertion, deletion or substitution of a     single amino acid residue on a position of the first sequence,     compared to the second sequence; it being understood that two amino     acid sequences can contain one, two or more such amino acid     differences; -   i) When a nucleotide sequence or amino acid sequence is said to     “comprise” another nucleotide sequence or amino acid sequence,     respectively, or to “essentially consist of” another nucleotide     sequence or amino acid sequence, this may mean that the latter     nucleotide sequence or amino acid sequence has been incorporated     into the first mentioned nucleotide sequence or amino acid sequence,     respectively, but more usually this generally means that the first     mentioned nucleotide sequence or amino acid sequence comprises     within its sequence a stretch of nucleotides or amino acid residues,     respectively, that has the same nucleotide sequence or amino acid     sequence, respectively, as the latter sequence, irrespective of how     the first mentioned sequence has actually been generated or obtained     (which may for example be by any suitable method described herein).     By means of a non-limiting example, when a Nanobody of the invention     is said to comprise a CDR sequence, this may mean that said CDR     sequence has been incorporated into the Nanobody of the invention,     but more usually this generally means that the Nanobody of the     invention contains within its sequence a stretch of amino acid     residues with the same amino acid sequence as said CDR sequence,     irrespective of how said Nanobody of the invention has been     generated or obtained. It should also be noted that when the latter     amino acid sequence has a specific biological or structural     function, it preferably has essentially the same, a similar or an     equivalent biological or structural function in the first mentioned     amino acid sequence (in other words, the first mentioned amino acid     sequence is preferably such that the latter sequence is capable of     performing essentially the same, a similar or an equivalent     biological or structural function). For example, when a Nanobody of     the invention is said to comprise a CDR sequence or framework     sequence, respectively, the CDR sequence and framework are     preferably capable, in said Nanobody, of functioning as a CDR     sequence or framework sequence, respectively. Also, when a     nucleotide sequence is said to comprise another nucleotide sequence,     the first mentioned nucleotide sequence is preferably such that,     when it is expressed into an expression product (e.g. a     polypeptide), the amino acid sequence encoded by the latter     nucleotide sequence forms part of said expression product (in other     words, that the latter nucleotide sequence is in the same reading     frame as the first mentioned, larger nucleotide sequence). -   j) A nucleic acid sequence or amino acid sequence is considered to     be “(in) essentially isolated (form)”—for example, compared to its     native biological source and/or the reaction medium or cultivation     medium from which it has been obtained—when it has been separated     from at least one other component with which it is usually     associated in said source or medium, such as another nucleic acid,     another protein/polypeptide, another biological component or     macromolecule or at least one contaminant, impurity or minor     component. In particular, a nucleic acid sequence or amino acid     sequence is considered “essentially isolated” when it has been     purified at least 2-fold, in particular at least 10-fold, more in     particular at least 100-fold, and up to 1000-fold or more. A nucleic     acid sequence or amino acid sequence that is “in essentially     isolated form” is preferably essentially homogeneous, as determined     using a suitable technique, such as a suitable chromatographical     technique, such as polyacrylamide-gel electrophoresis; -   k) The term “domain” as used herein generally refers to a globular     region of an amino acid sequence (such as an antibody chain, and in     particular to a globular region of a heavy chain antibody), or to a     polypeptide that essentially consists of such a globular region.     Usually, such a domain will comprise peptide loops (for example 3 or     4 peptide loops) stabilized, for example, as a sheet or by disulfide     bonds. The term “binding domain” refers to such a domain that is     directed against an antigenic determinant (as defined herein); -   l) The term “antigenic determinant” refers to the epitope on the     antigen recognized by the antigen-binding molecule (such as a     Nanobody or a polypeptide of the invention) and more in particular     by the antigen-binding site of said molecule. The terms “antigenic     determinant” and “epitope” may also be used interchangeably herein. -   m) An amino acid sequence (such as a Nanobody, an antibody, a     polypeptide of the invention, or generally an antigen binding     protein or polypeptide or a fragment thereof) that can     (specifically) bind to, that has affinity for and/or that has     specificity for a specific antigenic determinant, epitope, antigen     or protein (or for at least one part, fragment or epitope thereof)     is said to be “against” or “directed against” said antigenic     determinant, epitope, antigen or protein. -   n) The term “specificity” refers to the number of different types of     antigens or antigenic determinants to which a particular     antigen-binding molecule or antigen-binding protein (such as a     Nanobody or a polypeptide of the invention) molecule can bind. The     specificity of an antigen-binding protein can be determined based on     affinity and/or avidity. The affinity, represented by the     equilibrium constant for the dissociation of an antigen with an     antigen-binding protein (K_(D)), is a measure for the binding     strength between an antigenic determinant and an antigen-binding     site on the antigen-binding protein: the lesser the value of the     K_(D), the stronger the binding strength between an antigenic     determinant and the antigen-binding molecule (alternatively, the     affinity can also be expressed as the affinity constant (K_(A)),     which is 1/K_(D)). As will be clear to the skilled person (for     example on the basis of the further disclosure herein), affinity can     be determined in a manner known per se, depending on the specific     antigen of interest. Avidity is the measure of the strength of     binding between an antigen-binding molecule (such as a Nanobody or     polypeptide of the invention) and the pertinent antigen. Avidity is     related to both the affinity between an antigenic determinant and     its antigen binding site on the antigen-binding molecule and the     number of pertinent binding sites present on the antigen-binding     molecule. Typically, antigen-binding proteins (such as the amino     acid sequences, Nanobodies and/or polypeptides of the invention)     will bind to their antigen with a dissociation constant (K_(D)) of     10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²     moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter     (i.e. with an association constant (K_(A)) of 10⁵ to 10¹²     liter/moles or more, and preferably 10⁷ to 10¹² liter/moles or more     and more preferably 10⁸ to 10¹² liter/moles). Any K_(D) value     greater than 10⁴ mol/liter (or any K_(A) value lower than 10⁴ M⁻¹)     liters/mol is generally considered to indicate non-specific binding.     Preferably, a monovalent immunoglobulin sequence of the invention     will bind to the desired antigen with an affinity less than 500 nM,     preferably less than 200 nM, more preferably less than 10 nM, such     as less than 500 pM. Specific binding of an antigen-binding protein     to an antigen or antigenic determinant can be determined in any     suitable manner known per se, including, for example, Scatchard     analysis and/or competitive binding assays, such as     radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich     competition assays, and the different variants thereof known per se     in the art; as well as the other techniques mentioned herein.     -   The dissociation constant may be the actual or apparent         dissociation constant, as will be clear to the skilled person.         Methods for determining the dissociation constant will be clear         to the skilled person, and for example include the techniques         mentioned herein. In this respect, it will also be clear that it         may not be possible to measure dissociation constants of more         then 10⁻⁴ moles/liter or 10⁻³ moles/liter (e.g., of 10⁻²         moles/liter). Optionally, as will also be clear to the skilled         person, the (actual or apparent) dissociation constant may be         calculated on the basis of the (actual or apparent) association         constant (K_(A)), by means of the relationship [K_(D)=1/K_(A)].     -   The affinity denotes the strength or stability of a molecular         interaction. The affinity is commonly given as by the K_(D), or         dissociation constant, which has units of mol/liter (or M). The         affinity can also be expressed as an association constant,         K_(A), which equals 1/K_(D) and has units of (mol/liter)⁻¹ (or         M⁻¹). In the present specification, the stability of the         interaction between two molecules (such as an amino acid         sequence, Nanobody or polypeptide of the invention and its         intended target) will mainly be expressed in terms of the K_(D)         value of their interaction; it being clear to the skilled person         that in view of the relation K_(A)=1/K_(D), specifying the         strength of molecular interaction by its K_(D) value can also be         used to calculate the corresponding K_(A) value. The K_(D)-value         characterizes the strength of a molecular interaction also in a         thermodynamic sense as it is related to the free energy (DG) of         binding by the well known relation DG=RT.ln(K_(D)) (equivalently         DG=−RT.ln(K_(A))), where R equals the gas constant, T equals the         absolute temperature and In denotes the natural logarithm.     -   The K_(D) for biological interactions which are considered         meaningful (e.g. specific) are typically in the range of 10⁻¹⁰M         (0.1 nM) to 10⁻⁵M (10000 nM). The stronger an interaction is,         the lower is its K_(D).     -   The K_(D) can also be expressed as the ratio of the dissociation         rate constant of a complex, denoted as k_(off), to the rate of         its association, denoted k_(on) (so that K_(D)=k_(off)/k_(on)         and K_(A)=k_(on)/k_(off)). The off-rate k_(off) has units s⁻¹         (where s is the SI unit notation of second). The on-rate k_(on)         has units M⁻¹s⁻¹. The on-rate may vary between 10² M⁻¹s⁻¹ to         about 10⁷ M⁻¹s⁻¹, approaching the diffusion-limited association         rate constant for bimolecular interactions. The off-rate is         related to the half-life of a given molecular interaction by the         relation t_(1/2)=ln(2)/k_(off). The off-rate may vary between         10⁻⁶ s⁻¹ (near irreversible complex with a t_(1/2) of multiple         days) to 1 s⁻¹ (t_(1/2)=0.69 s).     -   The affinity of a molecular interaction between two molecules         can be measured via different techniques known per se, such as         the well known surface plasmon resonance (SPR) biosensor         technique (see for example Ober et al., Intern. Immunology, 13,         1551-1559, 2001) where one molecule is immobilized on the         biosensor chip and the other molecule is passed over the         immobilized molecule under flow conditions yielding k_(on),         k_(off) measurements and hence K_(D) (or K_(A)) values. This can         for example be performed using the well-known BIACORE         instruments.     -   It will also be clear to the skilled person that the measured         K_(D) may correspond to the apparent K_(D) if the measuring         process somehow influences the intrinsic binding affinity of the         implied molecules for example by artefacts related to the         coating on the biosensor of one molecule. Also, an apparent         K_(D) may be measured if one molecule contains more than one         recognition sites for the other molecule. In such situation the         measured affinity may be affected by the avidity of the         interaction by the two molecules.     -   Another approach that may be used to assess affinity is the         2-step ELISA (Enzyme-Linked Immunosorbent Assay) procedure of         Friguet et al. (J. Immunol. Methods, 77, 305-19, 1985). This         method establishes a solution phase binding equilibrium         measurement and avoids possible artefacts relating to adsorption         of one of the molecules on a support such as plastic.     -   However, the accurate measurement of K_(D) may be quite         labor-intensive and as consequence, often apparent K_(D) values         are determined to assess the binding strength of two molecules.         It should be noted that as long all measurements are made in a         consistent way (e.g. keeping the assay conditions unchanged)         apparent K_(D) measurements can be used as an approximation of         the true K_(D) and hence in the present document K_(D) and         apparent K_(D) should be treated with equal importance or         relevance. Finally, it should be noted that in many situations         the experienced scientist may judge it to be convenient to         determine the binding affinity relative to some reference         molecule. For example, to assess the binding strength between         molecules A and B, one may e.g. use a reference molecule C that         is known to bind to B and that is suitably labelled with a         fluorophore or chromophore group or other chemical moiety, such         as biotin for easy detection in an ELISA or FACS (Fluorescent         activated cell sorting) or other format (the fluorophore for         fluorescence detection, the chromophore for light absorption         detection, the biotin for streptavidin-mediated ELISA         detection). Typically, the reference molecule C is kept at a         fixed concentration and the concentration of A is varied for a         given concentration or amount of B. As a result an IC₅₀ value is         obtained corresponding to the concentration of A at which the         signal measured for C in absence of A is halved. Provided         K_(D ref), the K_(D) of the reference molecule, is known, as         well as the total concentration c_(ref) of the reference         molecule, the apparent K_(D) for the interaction A-B can be         obtained from following formula:         K_(D)=IC₅₀/(1+c_(ref)/K_(D ref)). Note that if         c_(ref)<<K_(D ref), K_(D)≈IC₅₀. Provided the measurement of the         IC₅₀ is performed in a consistent way (e.g. keeping c_(ref)         fixed) for the binders that are compared, the strength or         stability of a molecular interaction can be assessed by the IC₅₀         and this measurement is judged as equivalent to K_(D) or to         apparent K_(D) throughout this text. -   o) The half-life of an amino acid sequence, compound or polypeptide     of the invention can generally be defined as the time taken for the     serum concentration of the amino acid sequence, compound or     polypeptide to be reduced by 50%, in vivo, for example due to     degradation of the sequence or compound and/or clearance or     sequestration of the sequence or compound by natural mechanisms. The     in vivo half-life of an amino acid sequence, compound or polypeptide     of the invention can be determined in any manner known per se, such     as by pharmacokinetic analysis. Suitable techniques will be clear to     the person skilled in the art, and may for example generally involve     the steps of suitably administering to a warm-blooded animal (i.e.     to a human or to another suitable mammal, such as a mouse, rabbit,     rat, pig, dog or a primate, for example monkeys from the genus     Macaca (such as, and in particular, cynomolgus monkeys (Macaca     fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon     (Papio ursinus)) a suitable dose of the amino acid sequence,     compound or polypeptide of the invention; collecting blood samples     or other samples from said animal; determining the level or     concentration of the amino acid sequence, compound or polypeptide of     the invention in said blood sample; and calculating, from (a plot     of) the data thus obtained, the time until the level or     concentration of the amino acid sequence, compound or polypeptide of     the invention has been reduced by 50% compared to the initial level     upon dosing. Reference is for example made to the Experimental Part     below, as well as to the standard handbooks, such as Kenneth, A et     al: Chemical Stability of Pharmaceuticals: A Handbook for     Pharmacists and Peters et al, Pharmacokinete analysis: A Practical     Approach (1996). Reference is also made to “Pharmacokinetics”, M     Gibaldi & D Perron, published by Marcel Dekker, 2nd Rev. edition     (1982).     -   As will also be clear to the skilled person (see for example         pages 6 and 7 of WO 04/003019 and in the further references         cited therein), the half-life can be expressed using parameters         such as the t1/2-alpha, t1/2-beta and the area under the curve         (AUC). In the present specification, an “increase in half-life”         refers to an increase in any one of these parameters, such as         any two of these parameters, or essentially all three these         parameters. As used herein “increase in half-life” or “increased         half-life” in particular refers to an increase in the t1/2-beta,         either with or without an increase in the t1/2-alpha and/or the         AUC or both. -   p) In the context of the present invention, “modulating” or “to     modulate” generally means either reducing or inhibiting the activity     of, or alternatively increasing the activity of, a target or     antigen, as measured using a suitable in vitro, cellular or in vivo     assay. In particular, “modulating” or “to modulate” may mean either     reducing or inhibiting the activity of, or alternatively increasing     a (relevant or intended) biological activity of, a target or     antigen, as measured using a suitable in vitro, cellular or in vivo     assay (which will usually depend on the target or antigen involved),     by at least 1%, preferably at least 5%, such as at least 10% or at     least 25%, for example by at least 50%, at least 60%, at least 70%,     at least 80%, or 90% or more, compared to activity of the target or     antigen in the same assay under the same conditions but without the     presence of the construct of the invention.     -   As will be clear to the skilled person, “modulating” may also         involve effecting a change (which may either be an increase or a         decrease) in affinity, avidity, specificity and/or selectivity         of a target or antigen for one or more of its ligands, binding         partners, partners for association into a homomultimeric or         heteromultimeric form, or substrates; and/or effecting a change         (which may either be an increase or a decrease) in the         sensitivity of the target or antigen for one or more conditions         in the medium or surroundings in which the target or antigen is         present (such as pH, ion strength, the presence of co-factors,         etc.), compared to the same conditions but without the presence         of the construct of the invention. As will be clear to the         skilled person, this may again be determined in any suitable         manner and/or using any suitable assay known per se, depending         on the target or antigen involved.     -   “Modulating” may also mean effecting a change (i.e. an activity         as an agonist, as an antagonist or as a reverse agonist,         respectively, depending on the target or antigen and the desired         biological or physiological effect) with respect to one or more         biological or physiological mechanisms, effects, responses,         functions, pathways or activities in which the target or antigen         (or in which its substrate(s), ligand(s) or pathway(s) are         involved, such as its signalling pathway or metabolic pathway         and their associated biological or physiological effects) is         involved. Again, as will be clear to the skilled person, such an         action as an agonist or an antagonist may be determined in any         suitable manner and/or using any suitable (in vitro and usually         cellular or in assay) assay known per se, depending on the         target or antigen involved. In particular, an action as an         agonist or antagonist may be such that an intended biological or         physiological activity is increased or decreased, respectively,         by at least 1%, preferably at least 5%, such as at least 10% or         at least 25%, for example by at least 50%, at least 60%, at         least 70%, at least 80%, or 90% or more, compared to the         biological or physiological activity in the same assay under the         same conditions but without the presence of the construct of the         invention.     -   Modulating may for example also involve allosteric modulation of         the target or antigen; and/or reducing or inhibiting the binding         of the target or antigen to one of its substrates or ligands         and/or competing with a natural ligand, substrate for binding to         the target or antigen. Modulating may also involve activating         the target or antigen or the mechanism or pathway in which it is         involved. Modulating may for example also involve effecting a         change in respect of the folding or confirmation of the target         or antigen, or in respect of the ability of the target or         antigen to fold, to change its confirmation (for example, upon         binding of a ligand), to associate with other (sub)units, or to         disassociate. Modulating may for example also involve effecting         a change in the ability of the target or antigen to transport         other compounds or to serve as a channel for other compounds         (such as ions).     -   Modulating may be reversible or irreversible, but for         pharmaceutical and pharmacological purposes will usually be in a         reversible manner. -   q) In respect of a target or antigen, the term “interaction site” on     the target or antigen means a site, epitope, antigenic determinant,     part, domain or stretch of amino acid residues on the target or     antigen that is a site for binding to a ligand, receptor or other     binding partner, a catalytic site, a cleavage site, a site for     allosteric interaction, a site involved in multimerisation (such as     homomerization or heterodimerization) of the target or antigen; or     any other site, epitope, antigenic determinant, part, domain or     stretch of amino acid residues on the target or antigen that is     involved in a biological action or mechanism of the target or     antigen. More generally, an “interaction site” can be any site,     epitope, antigenic determinant, part, domain or stretch of amino     acid residues on the target or antigen to which an amino acid     sequence or polypeptide of the invention can bind such that the     target or antigen (and/or any pathway, interaction, signalling,     biological mechanism or biological effect in which the target or     antigen is involved) is modulated (as defined herein). -   r) An amino acid sequence or polypeptide is said to be “specific     for” a first target or antigen compared to a second target or     antigen when is binds to the first antigen with an affinity (as     described above, and suitably expressed as a K_(D) value, K_(A)     value, K_(off) rate and/or K_(on) rate) that is at least 10 times,     such as at least 100 times, and preferably at least 1000 times, and     up to 10.000 times or more better than the affinity with which said     amino acid sequence or polypeptide binds to the second target or     polypeptide. For example, the first antigen may bind to the target     or antigen with a K_(D) value that is at least 10 times less, such     as at least 100 times less, and preferably at least 1000 times less,     such as 10.000 times less or even less than that, than the K_(D)     with which said amino acid sequence or polypeptide binds to the     second target or polypeptide.     -   Preferably, when an amino acid sequence or polypeptide is         “specific for” a first target or antigen compared to a second         target or antigen, it is directed against (as defined herein)         said first target or antigen, but not directed against said         second target or antigen. -   s) The terms “cross-block”, “cross-blocked” and “cross-blocking” are     used interchangeably herein to mean the ability of an amino acid     sequence or other binding agents (such as a polypeptide of the     invention) to interfere with the binding of other amino acid     sequences or binding agents of the invention to a given target. The     extend to which an amino acid sequence or other binding agents of     the invention is able to interfere with the binding of another to     [target], and therefore whether it can be said to cross-block     according to the invention, can be determined using competition     binding assays. One particularly suitable quantitative assay uses a     Biacore machine which can measure the extent of interactions using     surface plasmon resonance technology. Another suitable quantitative     cross-blocking assay uses an ELISA-based approach to measure     competition between amino acid sequence or another binding agents in     terms of their binding to the target.     -   The following generally describes a suitable Biacore assay for         determining whether an amino acid sequence or other binding         agent cross-blocks or is capable of cross-blocking according to         the invention. It will be appreciated that the assay can be used         with any of the amino acid sequence or other binding agents         described herein. The Biacore machine (for example the         Biacore 3000) is operated in line with the manufacturer's         recommendations. Thus in one cross-blocking assay, the target         protein is coupled to a CM5 Biacore chip using standard amine         coupling chemistry to generate a surface that is coated with the         target. Typically 200-800 resonance units of the target would be         coupled to the chip (an amount that gives easily measurable         levels of binding but that is readily saturable by the         concentrations of test reagent being used). Two test amino acid         sequences (termed A* and B*) to be assessed for their ability to         cross-block each other are mixed at a one to one molar ratio of         binding sites in a suitable buffer to create the test mixture.         When calculating the concentrations on a binding site basis the         molecular weight of an amino acid sequence is assumed to be the         total molecular weight of the amino acid sequence divided by the         number of target binding sites on that amino acid sequence. The         concentration of each amino acid sequence in the test mix should         be high enough to readily saturate the binding sites for that         amino acid sequence on the target molecules captured on the         Biacore chip. The amino acid sequences in the mixture are at the         same molar concentration (on a binding basis) and that         concentration would typically be between 1.00 and 1.5 micromolar         (on a binding site basis). Separate solutions containing A*         alone and B* alone are also prepared. A* and B* in these         solutions should be in the same buffer and at the same         concentration as in the test mix. The test mixture is passed         over the target-coated Biacore chip and the total amount of         binding recorded. The chip is then treated in such a way as to         remove the bound amino acid sequences without damaging the         chip-bound target. Typically this is done by treating the chip         with 30 mM HCl for 60 seconds. The solution of A* alone is then         passed over the target-coated surface and the amount of binding         recorded. The chip is again treated to remove all of the bound         amino acid sequences without damaging the chip-bound target. The         solution of B* alone is then passed over the target-coated         surface and the amount of binding recorded. The maximum         theoretical binding of the mixture of A* and B* is next         calculated, and is the sum of the binding of each amino acid         sequence when passed over the target surface alone. If the         actual recorded binding of the mixture is less than this         theoretical maximum then the two amino acid sequences are         cross-blocking each other. Thus, in general, a cross-blocking         amino acid sequence or other binding agent according to the         invention is one which will bind to the target in the above         Biacore cross-blocking assay such that during the assay and in         the presence of a second amino acid sequence or other binding         agent of the invention the recorded binding is between 80% and         0.1% (e.g. 80% to 4%) of the maximum theoretical binding,         specifically between 75% and 0.1% (e.g. 75% to 4%) of the         maximum theoretical binding, and more specifically between 70%         and 0.1% (e.g. 70% to 4%) of maximum theoretical binding (as         just defined above) of the two amino acid sequences or binding         agents in combination. The Biacore assay described above is a         primary assay used to determine if amino acid sequences or other         binding agents cross-block each other according to the         invention. On rare occasions particular amino acid sequences or         other binding agents may not bind to target coupled via amine         chemistry to a CM5 Biacore chip (this usually occurs when the         relevant binding site on target is masked or destroyed by the         coupling to the chip). In such cases cross-blocking can be         determined using a tagged version of the target, for example a         N-terminal His-tagged version (R & D Systems, Minneapolis,         Minn., USA; 2005 cat #1406-ST-025). In this particular format,         an anti-His amino acid sequence would be coupled to the Biacore         chip and then the His-tagged target would be passed over the         surface of the chip and captured by the anti-His amino acid         sequence. The cross blocking analysis would be carried out         essentially as described above, except that after each chip         regeneration cycle, new His-tagged target would be loaded back         onto the anti-His amino acid sequence coated surface. In         addition to the example given using N-terminal His-tagged         [target], C-terminal His-tagged target could alternatively be         used. Furthermore, various other tags and tag binding protein         combinations that are known in the art could be used for such a         cross-blocking analysis (e.g. HA tag with anti-HA antibodies;         FLAG tag with anti-FLAG antibodies; biotin tag with         streptavidin).     -   The following generally describes an ELISA assay for determining         whether an amino acid sequence or other binding agent directed         against a target cross-blocks or is capable of cross-blocking as         defined herein. It will be appreciated that the assay can be         used with any of the amino acid sequences (or other binding         agents such as polypeptides of the invention) described herein.         The general principal of the assay is to have an amino acid         sequence or binding agent that is directed against the target         coated onto the wells of an ELISA plate. An excess amount of a         second, potentially cross-blocking, anti-target amino acid         sequence is added in solution (i.e. not bound to the ELISA         plate). A limited amount of the target is then added to the         wells. The coated amino acid sequence and the amino acid         sequence in solution compete for binding of the limited number         of target molecules. The plate is washed to remove excess target         that has not been bound by the coated amino acid sequence and to         also remove the second, solution phase amino acid sequence as         well as any complexes formed between the second, solution phase         amino acid sequence and target. The amount of bound target is         then measured using a reagent that is appropriate to detect the         target. An amino acid sequence in solution that is able to         cross-block the coated amino acid sequence will be able to cause         a decrease in the number of target molecules that the coated         amino acid sequence can bind relative to the number of target         molecules that the coated amino acid sequence can bind in the         absence of the second, solution phase, amino acid sequence. In         the instance where the first amino acid sequence, e.g. an Ab-X,         is chosen to be the immobilized amino acid sequence, it is         coated onto the wells of the ELISA plate, after which the plates         are blocked with a suitable blocking solution to minimize         non-specific binding of reagents that are subsequently added. An         excess amount of the second amino acid sequence, i.e. Ab-Y, is         then added to the ELISA plate such that the moles of Ab-Y         [target] binding sites per well are at least 10 fold higher than         the moles of Ab-X [target] binding sites that were used, per         well, during the coating of the ELISA plate. [target] is then         added such that the moles of [target] added per well are at         least 25-fold lower than the moles of Ab-X [target] binding         sites that were used for coating each well. Following a suitable         incubation period the ELISA plate is washed and a reagent for         detecting the target is added to measure the amount of target         specifically bound by the coated anti-[target] amino acid         sequence (in this case Ab-X). The background signal for the         assay is defined as the signal obtained in wells with the coated         amino acid sequence (in this case Ab-X), second solution phase         amino acid sequence (in this case Ab-Y), [target] buffer only         (i.e. no target) and target detection reagents. The positive         control signal for the assay is defined as the signal obtained         in wells with the coated amino acid sequence (in this case         Ab-X), second solution phase amino acid sequence buffer only         (i.e. no second solution phase amino acid sequence), target and         target detection reagents. The ELISA assay may be run in such a         manner so as to have the positive control signal be at least 6         times the background signal. To avoid any artefacts (e.g.         significantly different affinities between Ab-X and Ab-Y for         [target]) resulting from the choice of which amino acid sequence         to use as the coating amino acid sequence and which to use as         the second (competitor) amino acid sequence, the cross-blocking         assay may to be run in two formats: 1) format 1 is where Ab-X is         the amino acid sequence that is coated onto the ELISA plate and         Ab-Y is the competitor amino acid sequence that is in solution         and 2) format 2 is where Ab-Y is the amino acid sequence that is         coated onto the ELISA plate and Ab-X is the competitor amino         acid sequence that is in solution. Ab-X and Ab-Y are defined as         cross-blocking if, either in format 1 or in format 2, the         solution phase anti-target amino acid sequence is able to cause         a reduction of between 60% and 100%, specifically between 70%         and 100%, and more specifically between 80% and 100%, of the         target detection signal {i.e. the amount of target bound by the         coated amino acid sequence) as compared to the target detection         signal obtained in the absence of the solution phase anti-target         amino acid sequence (i.e. the positive control wells). -   t) As further described herein, the total number of amino acid     residues in a Nanobody can be in the region of 110-120, is     preferably 112-115, and is most preferably 113. It should however be     noted that parts, fragments, analogs or derivatives (as further     described herein) of a Nanobody are not particularly limited as to     their length and/or size, as long as such parts, fragments, analogs     or derivatives meet the further requirements outlined herein and are     also preferably suitable for the purposes described herein; -   u) The amino acid residues of a Nanobody are numbered according to     the general numbering for V_(H) domains given by Kabat et al.     (“Sequence of proteins of immunological interest”, US Public Health     Services, NIH Bethesda, Md., Publication No. 91), as applied to     V_(HH) domains from Camelids in the article of Riechmann and     Muyldermans, J. Immunol. Methods Jun. 23, 2000; 240 (1-2): 185-195     (see for example FIG. 2 of this publication); or referred to herein.     According to this numbering, FR1 of a Nanobody comprises the amino     acid residues at positions 1-30, CDR1 of a Nanobody comprises the     amino acid residues at positions 31-35, FR2 of a Nanobody comprises     the amino acids at positions 36-49, CDR2 of a Nanobody comprises the     amino acid residues at positions 50-65, FR3 of a Nanobody comprises     the amino acid residues at positions 66-94, CDR3 of a Nanobody     comprises the amino acid residues at positions 95-102, and FR4 of a     Nanobody comprises the amino acid residues at positions 103-113. [In     this respect, it should be noted that—as is well known in the art     for V_(H) domains and for V_(HH) domains—the total number of amino     acid residues in each of the CDR's may vary and may not correspond     to the total number of amino acid residues indicated by the Kabat     numbering (that is, one or more positions according to the Kabat     numbering may not be occupied in the actual sequence, or the actual     sequence may contain more amino acid residues than the number     allowed for by the Kabat numbering). This means that, generally, the     numbering according to Kabat may or may not correspond to the actual     numbering of the amino acid residues in the actual sequence.     Generally, however, it can be said that, according to the numbering     of Kabat and irrespective of the number of amino acid residues in     the CDR's, position 1 according to the Kabat numbering corresponds     to the start of FR1 and vice versa, position 36 according to the     Kabat numbering corresponds to the start of FR2 and vice versa,     position 66 according to the Kabat numbering corresponds to the     start of FR3 and vice versa, and position 103 according to the Kabat     numbering corresponds to the start of FR4 and vice versa.].     -   Alternative methods for numbering the amino acid residues of         V_(H) domains, which methods can also be applied in an analogous         manner to V_(HH) domains from Camelids and to Nanobodies, are         the method described by Chothia et al. (Nature 342, 877-883         (1989)), the so-called “AbM definition” and the so-called         “contact definition”. However, in the present description,         claims and figures, the numbering according to Kabat as applied         to V_(HH) domains by Riechmann and Muyldermans will be followed,         unless indicated otherwise; and -   v) The Figures, Sequence Listing and the Experimental Part/Examples     are only given to further illustrate the invention and should not be     interpreted or construed as limiting the scope of the invention     and/or of the appended claims in any way, unless explicitly     indicated otherwise herein.

For a general description of heavy chain antibodies and the variable domains thereof, reference is inter alia made to the prior art cited herein, to the review article by Muyldermans in Reviews in Molecular Biotechnology 74(2001), 277-302; as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 by the National Research Council of Canada; WO 03/025020 (=EP 1 433 793) by the Institute of Antibodies; as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO 06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V. Reference is also made to the further prior art mentioned in these applications, and in particular to the list of references mentioned on pages 41-43 of the International application WO 06/040153, which list and references are incorporated herein by reference.

In accordance with the terminology used in the art (see the above references), the variable domains present in naturally occurring heavy chain antibodies will also be referred to as “V_(HH) domains”, in order to distinguish them from the heavy chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as “V_(H) domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which will be referred to hereinbelow as “V_(L) domains”).

As mentioned in the prior art referred to above, V_(HH) domains have a number of unique structural characteristics and functional properties which make isolated V_(HH) domains (as well as Nanobodies based thereon, which share these structural characteristics and functional properties with the naturally occurring V_(HH) domains) and proteins containing the same highly advantageous for use as functional antigen-binding domains or proteins. In particular, and without being limited thereto, V_(HH) domains (which have been “designed” by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) and Nanobodies can function as a single, relatively small, functional antigen-binding structural unit, domain or protein. This distinguishes the V_(HH) domains from the V_(H) and V_(L) domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigen-binding proteins or domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in ScFv's fragments, which consist of a V_(H) domain covalently linked to a V_(L) domain).

Because of these unique properties, the use of V_(HH) domains and Nanobodies as single antigen-binding proteins or as antigen binding domains (i.e. as part of a larger protein or polypeptide) offers a number of significant advantages over the use of conventional V_(H) and V_(L) domains, scFv's or conventional antibody fragments (such as Fab- or F(ab′)₂-fragments):

-   -   only a single domain is required to bind an antigen with high         affinity and with high selectivity, so that there is no need to         have two separate domains present, nor to assure that these two         domains are present in the right spatial conformation and         configuration (i.e. through the use of especially designed         linkers, as with scFv's);     -   V_(HH) domains and Nanobodies can be expressed from a single         gene and require no post-translational folding or modifications;     -   V_(HH) domains and Nanobodies can easily be engineered into         multivalent and multispecific formats (as further discussed         herein);     -   V_(HH) domains and Nanobodies are highly soluble and do not have         a tendency to aggregate (as with the mouse-derived “dAb's”         described by Ward et al., Nature, Vol. 341, 1989, p. 544);     -   V_(HH) domains and Nanobodies are highly stable to heat, pH,         proteases and other denaturing agents or conditions (see for         example Ewert et al, supra);     -   V_(HH) domains and Nanobodies are easy and relatively cheap to         prepare, even on a scale required for production. For example,         V_(HH) domains, Nanobodies and proteins/polypeptides containing         the same can be produced using microbial fermentation (e.g. as         further described below) and do not require the use of mammalian         expression systems, as with for example conventional antibody         fragments;     -   V_(HH) domains and Nanobodies are relatively small         (approximately 15 kDa, or 10 times smaller than a conventional         IgG) compared to conventional 4-chain antibodies and         antigen-binding fragments thereof, and therefore show high(er)         penetration into tissues (including but not limited to solid         tumors and other dense tissues) than such conventional 4-chain         antibodies and antigen-binding fragments thereof;     -   V_(HH) domains and Nanobodies can show so-called cavity-binding         properties (inter alia due to their extended CDR3 loop, compared         to conventional V_(H) domains) and can therefore also access         targets and epitopes not accessible to conventional 4-chain         antibodies and antigen-binding fragments thereof. For example,         it has been shown that V_(HH) domains and Nanobodies can inhibit         enzymes (see for example WO 97/49805; Transue et al., Proteins         Sep. 1, 1998; 32(4): 515-22; Lauwereys et al., EMBO J. Jul. 1,         1998; 17(13): 3512-20).

In a specific and preferred aspect, the invention provides Nanobodies against a metalloproteinase from the ADAM family, and in particular Nanobodies against a metalloproteinase from the ADAM family from a warm-blooded animal, and more in particular Nanobodies against a metalloproteinase from the ADAM family from a mammal, and especially Nanobodies against human a metalloproteinase from the ADAM family; as well as proteins and/or polypeptides comprising at least one such Nanobody.

In particular, the invention provides Nanobodies against a metalloproteinase from the ADAM family, and proteins and/or polypeptides comprising the same, that have improved therapeutic and/or pharmacological properties and/or other advantageous properties (such as, for example, improved ease of preparation and/or reduced costs of goods), compared to conventional antibodies against a metalloproteinase from the ADAM family or fragments thereof, compared to constructs that could be based on such conventional antibodies or antibody fragments (such as Fab′ fragments, F(ab′)₂ fragments, ScFv constructs, “diabodies” and other multispecific constructs (see for example the review by Holliger and Hudson, Nat Biotechnol. September 2005; 23(9):1126-36)), and also compared to the so-called “dAb's” or similar (single) domain antibodies that may be derived from variable domains of conventional antibodies. These improved and advantageous properties will become clear from the further description herein, and for example include, without limitation, one or more of:

-   -   increased affinity and/or avidity for a metalloproteinase from         the ADAM family, either in a monovalent format, in a multivalent         format (for example in a bivalent format) and/or in a         multispecific format (for example one of the multispecific         formats described hereinbelow);     -   better suitability for formatting in a multivalent format (for         example in a bivalent format);     -   better suitability for formatting in a multispecific format (for         example one of the multispecific formats described hereinbelow);     -   improved suitability or susceptibility for “humanizing”         substitutions (as defined herein);     -   less immunogenicity, either in a monovalent format, in a         multivalent format (for example in a bivalent format) and/or in         a multispecific format (for example one of the multispecific         formats described hereinbelow);     -   increased stability, either in a monovalent format, in a         multivalent format (for example in a bivalent format) and/or in         a multispecific format (for example one of the multispecific         formats described hereinbelow);     -   increased specificity towards a metalloproteinase from the ADAM         family, either in a monovalent format, in a multivalent format         (for example in a bivalent format) and/or in a multispecific         format (for example one of the multispecific formats described         hereinbelow);     -   decreased or where desired increased cross-reactivity with a         metalloproteinase from the ADAM family from different species;         and/or     -   one or more other improved properties desirable for         pharmaceutical use (including prophylactic use and/or         therapeutic use) and/or for diagnostic use (including but not         limited to use for imaging purposes), either in a monovalent         format, in a multivalent format (for example in a bivalent         format) and/or in a multispecific format (for example one of the         multispecific formats described hereinbelow).

As generally described herein for the amino acid sequences of the invention, the Nanobodies of the invention are preferably in essentially isolated form (as defined herein), or form part of a protein or polypeptide of the invention (as defined herein), which may comprise or essentially consist of one or more Nanobodies of the invention and which may optionally further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). For example, and without limitation, the one or more amino acid sequences of the invention may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit (i.e. against one or more other targets than a metalloproteinase from the ADAM family), so as to provide a monovalent, multivalent or multispecific polypeptide of the invention, respectively, all as described herein. In particular, such a protein or polypeptide may comprise or essentially consist of one or more Nanobodies of the invention and optionally one or more (other) Nanobodies (i.e. directed against other targets than a metalloproteinase from the ADAM family), all optionally linked via one or more suitable linkers, so as to provide a monovalent, multivalent or multispecific Nanobody construct, respectively, as further described herein. Such proteins or polypeptides may also be in essentially isolated form (as defined herein).

In a Nanobody of the invention, the binding site for binding against a metalloproteinase from the ADAM family is preferably formed by the CDR sequences. Optionally, a Nanobody of the invention may also, and in addition to the at least one binding site for binding against a metalloproteinase from the ADAM family, contain one or more further binding sites for binding against other antigens, proteins or targets. For methods and positions for introducing such second binding sites, reference is for example made to Keck and Huston, Biophysical Journal, 71, October 1996, 2002-2011; EP 0 640 130 and WO 06/07260.

As generally described herein for the amino acid sequences of the invention, when a Nanobody of the invention (or a polypeptide of the invention comprising the same) is intended for administration to a subject (for example for therapeutic and/or diagnostic purposes as described herein), it is preferably directed against human a metalloproteinase from the ADAM family; whereas for veterinary purposes, it is preferably directed against a metalloproteinase from the ADAM family from the species to be treated. Also, as with the amino acid sequences of the invention, a Nanobody of the invention may or may not be cross-reactive (i.e. directed against a metalloproteinase from the ADAM family from two or more species of mammal, such as against human a metalloproteinase from the ADAM family and a metalloproteinase from the ADAM family from at least one of the species of mammal mentioned herein).

Also, again as generally described herein for the amino acid sequences of the invention, the Nanobodies of the invention may generally be directed against any antigenic determinant, epitope, part, domain, subunit or confirmation (where applicable) of a metalloproteinase from the ADAM family. However, it is generally assumed and preferred that the Nanobodies of the invention (and polypeptides comprising the same) are directed against one of the catalytic sites or binding sites mentioned generally herein for the amino acid sequences of the invention.

As already described herein, the amino acid sequence and structure of a Nanobody can be considered—without however being limited thereto—to be comprised of four framework regions or “FR's” (or sometimes also referred to as “FW's”), which are referred to in the art and herein as “Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as “Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”, respectively; which framework regions are interrupted by three complementary determining regions or “CDR's”, which are referred to in the art as “Complementarity Determining Region 1” or “CDR1”; as “Complementarity Determining Region 2” or “CDR2”; and as “Complementarity Determining Region 3” or “CDR3”, respectively. Some preferred framework sequences and CDR's (and combinations thereof) that are present in the Nanobodies of the invention are as described herein. Other suitable CDR sequences can be obtained by the methods described herein.

According to a non-limiting but preferred aspect of the invention, (the CDR sequences present in) the Nanobodies of the invention are such that:

-   -   the Nanobodies can bind to a metalloproteinase from the ADAM         family with a dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹²         moles/liter or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or         less and more preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an         association constant (K_(A)) of 10⁵ to 10¹² liter/moles or more,         and preferably 10⁷ to 10¹² liter/moles or more and more         preferably 10⁸ to 10¹² liter/moles);         and/or such that:     -   the Nanobodies can bind to a metalloproteinase from the ADAM         family with a k_(on)-rate of between 10² M⁻¹s⁻¹ to about 10⁷         M⁻¹s⁻¹, preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more         preferably between 10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between         10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;         and/or such that they:     -   the Nanobodies can bind to a metalloproteinase from the ADAM         family with a k_(off) rate between 1 s⁻¹ (t_(1/2)=0.69 s) and         10⁻⁶ s⁻¹ (providing a near irreversible complex with a t_(1/2)         of multiple days), preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹,         more preferably between 10⁻³ s⁻¹ and 10⁻⁴ s⁻¹, such as between         10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Preferably, (the CDR sequences present in) the Nanobodies of the invention are such that: a monovalent Nanobody of the invention (or a polypeptide that contains only one Nanobody of the invention) is preferably such that it will bind to a metalloproteinase from the ADAM family with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

The affinity of the Nanobody of the invention against a metalloproteinase from the ADAM family can be determined in a manner known per se, for example using the general techniques for measuring K_(D). K_(A), k_(off) or k_(on) mentioned herein, as well as some of the specific assays described herein.

Some preferred IC50 values for binding of the Nanobodies of the invention (and of polypeptides comprising the same) to a metalloproteinase from the ADAM family will become clear from the further description and examples herein.

In a preferred but non-limiting aspect, the invention relates to a Nanobody (as defined herein) against a metalloproteinase from the ADAM family, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     337;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     337;     and/or     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 549;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     549;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     549;     and/or     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 761;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     761;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     761;     or any suitable fragment of such an amino acid sequence.

In particular, according to this preferred but non-limiting aspect, the invention relates to a Nanobody (as defined herein) against a metalloproteinase from the ADAM family, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 337;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     337;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     337;     and     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 549;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     549;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     549;     and     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 761;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     761;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     761;     or any suitable fragment of such an amino acid sequences.

In a specific but non-limiting aspect, the invention relates to a Nanobody (as defined herein) against a ADAMS, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     250;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     250;     and/or     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 462;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     462;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     462;     and/or     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 674;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     674;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     674;     or any suitable fragment of such an amino acid sequence.

In particular, according to this specific but non-limiting aspect, the invention relates to a Nanobody (as defined herein) ADAMS, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 232 to 250;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     250;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 232 to     250;     and     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 444 to 462;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     462;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 444 to     462;     and     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 656 to 674;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     674;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 656 to     674;     or any suitable fragment of such an amino acid sequences.

In a specific but non-limiting aspect, the invention relates to a Nanobody (as defined herein) against a ADAM9, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to     987;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 251 to     271 and/or 984 to 987;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 251 to     271 and/or 984 to 987;     and/or     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004     to 1007;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 463 to     483 and/or 1004 to 1007;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 463 to     483 and/or 1004 to 1007;     and/or     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024     to 1027;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 675 to     695 and/or 1024 to 1027;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 675 to     695 and/or 1024 to 1027;     or any suitable fragment of such an amino acid sequence.

In particular, according to this specific but non-limiting aspect, the invention relates to a Nanobody (as defined herein) ADAM9, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 251 to 271 and/or 984 to     987;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 251 to     271 and/or 984 to 987;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 251 to     271 and/or 984 to 987;     and     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 463 to 483 and/or 1004     to 1007;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 463 to     483 and/or 1004 to 1007;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 463 to     483 and/or 1004 to 1007;     and     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 675 to 695 and/or 1024     to 1027;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 675 to     695 and/or 1024 to 1027;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 675 to     695 and/or 1024 to 1027;     or any suitable fragment of such an amino acid sequences.

In a specific but non-limiting aspect, the invention relates to a Nanobody (as defined herein) against a ADAM10, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 272 to     295;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 272 to     295;     and/or     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 484 to 507;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 484 to     507;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 484 to     507;     and/or     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 696 to 719;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 696 to     719;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 696 to     719;     or any suitable fragment of such an amino acid sequence.

In particular, according to this specific but non-limiting aspect, the invention relates to a Nanobody (as defined herein) ADAM10, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 272 to 295;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 272 to     295;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 272 to     295;     and     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 484 to 507;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 484 to     507;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 484 to     507;     and     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 696 to 719;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 696 to     719;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 696 to     719;     or any suitable fragment of such an amino acid sequences.

In a specific but non-limiting aspect, the invention relates to a Nanobody (as defined herein) against a ADAM17, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to     992;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 296 to     312 and/or 988 to 992;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 296 to     312 and/or 988 to 992;     and/or     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008     to 1012;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 508 to     524 and/or 1008 to 1012;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 508 to     524 and/or 1008 to 1012;     and/or     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028     to 1032;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 720 to     736 and/or 1028 to 1032;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 720 to     736 and/or 1028 to 1032;     or any suitable fragment of such an amino acid sequence.

In particular, according to this specific but non-limiting aspect, the invention relates to a Nanobody (as defined herein) ADAM17, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 296 to 312 and/or 988 to     992;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 296 to     312 and/or 988 to 992;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 296 to     312 and/or 988 to 992;     and     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 508 to 524 and/or 1008     to 1012;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 508 to     524 and/or 1008 to 1012;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 508 to     524 and/or 1008 to 1012;     and     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 720 to 736 and/or 1028     to 1032;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 720 to     736 and/or 1028 to 1032;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 720 to     736 and/or 1028 to 1032;     or any suitable fragment of such an amino acid sequences.

In a specific but non-limiting aspect, the invention relates to a Nanobody (as defined herein) against a ADAMTS5, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 and/or 993;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 313 to     337 and/or 993;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 313 to     337 and/or 993;     and/or     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 525 to 549 and/or 1013;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 525 to     549 and/or 1013;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 525 to     549 and/or 1013;     and/or     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 737 to 761 and/or 1033;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 737 to     761 and/or 1033;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 737 to     761 and/or 1033;     or any suitable fragment of such an amino acid sequence.

In particular, according to this specific but non-limiting aspect, the invention relates to a Nanobody (as defined herein) ADAMTS5, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which:

-   -   CDR1 is chosen from the group consisting of:

-   a) the amino acid sequences of SEQ ID NO's: 313 to 337 and/or 993;

-   b) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 313 to     337 and/or 993;

-   c) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 313 to     337 and/or 993;     and     -   CDR2 is chosen from the group consisting of:

-   d) the amino acid sequences of SEQ ID NO's: 525 to 549 and/or 1013;

-   e) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 525 to     549 and/or 1013;

-   f) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 525 to     549 and/or 1013;     and     -   CDR3 is chosen from the group consisting of:

-   g) the amino acid sequences of SEQ ID NO's: 737 to 761 and/or 1033;

-   h) amino acid sequences that have at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 737 to     761 and/or 1033;

-   i) amino acid sequences that have 3, 2, or 1 amino acid difference     with at least one of the amino acid sequences of SEQ ID NO's: 737 to     761 and/or 1033;     or any suitable fragment of such an amino acid sequences.

As generally mentioned herein for the amino acid sequences of the invention, when one of the Nanobody of the invention mentioned in one of the preceding paragraphs contains one or more CDR1 sequences according to b) and/or c):

-   i) any amino acid substitution in such a CDR according to b)     and/or c) is preferably, and compared to the corresponding CDR     according to a), a conservative amino acid substitution (as defined     herein);     and/or -   ii) the CDR according to b) and/or c) preferably only contains amino     acid substitutions, and no amino acid deletions or insertions,     compared to the corresponding CDR according to a);     and/or -   iii) the CDR according to b) and/or c) may be a CDR that is derived     from a CDR according to a) by means of affinity maturation using one     or more techniques of affinity maturation known per se.

Similarly, when one of the Nanobody of the invention mentioned in one of the preceding paragraphs contains one or more CDR2 sequences according to e) and/or f):

-   i) any amino acid substitution in such a CDR according to e)     and/or f) is preferably, and compared to the corresponding CDR     according to d), a conservative amino acid substitution (as defined     herein);     and/or -   ii) the CDR according to e) and/or f) preferably only contains amino     acid substitutions, and no amino acid deletions or insertions,     compared to the corresponding CDR according to d);     and/or -   iii) the CDR according to e) and/or f) may be a CDR that is derived     from a CDR according to d) by means of affinity maturation using one     or more techniques of affinity maturation known per se.

Also, similarly, when one of the Nanobody of the invention mentioned in one of the preceding paragraphs contains one or more CDR3 sequences according to h) and/or i):

-   i) any amino acid substitution in such a CDR according to h)     and/or i) is preferably, and compared to the corresponding CDR     according to g), a conservative amino acid substitution (as defined     herein);     and/or -   ii) the CDR according to h) and/or i) preferably only contains amino     acid substitutions, and no amino acid deletions or insertions,     compared to the corresponding CDR according to g);     and/or -   iii) the CDR according to h) and/or i) may be a CDR that is derived     from a CDR according to g) by means of affinity maturation using one     or more techniques of affinity maturation known per se.

It should be understood that the last three paragraphs generally apply to any Nanobody of the invention that comprises one or more CDR1 sequences, CDR2 sequences and/or CDR3 sequences according to b), c), e), f), h) or i), respectively.

Of the Nanobodies of the invention, Nanobodies comprising one or more of the CDR's explicitly listed above are particularly preferred; Nanobodies comprising two or more of the CDR's explicitly listed above are more particularly preferred; and Nanobodies comprising three of the CDR's explicitly listed above are most particularly preferred.

Some particularly preferred, but non-limiting combinations of CDR sequences, as well as preferred combinations of CDR sequences and framework sequences, are mentioned in Table A-1 below, which lists the CDR sequences and framework sequences that are present in a number of preferred (but non-limiting) Nanobodies of the invention. As will be clear to the skilled person, a combination of CDR1, CDR2 and CDR3 sequences that occur in the same clone (i.e. CDR1, CDR2 and CDR3 sequences that are mentioned on the same line in Table A-1) will usually be preferred (although the invention in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences mentioned in Table A-1). Also, a combination of CDR sequences and framework sequences that occur in the same clone (i.e. CDR sequences and framework sequences that are mentioned on the same line in Table A-1) will usually be preferred (although the invention in its broadest sense is not limited thereto, and also comprises other suitable combinations of the CDR sequences and framework sequences mentioned in Table A-1, as well as combinations of such CDR sequences and other suitable framework sequences, e.g. as further described herein).

Also, in the Nanobodies of the invention that comprise the combinations of CDR's mentioned in Table A-1, each CDR can be replaced by a CDR chosen from the group consisting of amino acid sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the mentioned CDR's; in which:

-   i) any amino acid substitution in such a CDR is preferably, and     compared to the corresponding CDR sequence mentioned in Table A-1, a     conservative amino acid substitution (as defined herein);     and/or -   ii) any such CDR sequence preferably only contains amino acid     substitutions, and no amino acid deletions or insertions, compared     to the corresponding CDR sequence mentioned in Table A-1;     and/or -   iii) any such CDR sequence is a CDR that is derived by means of a     technique for affinity maturation known per se, and in particular     starting from the corresponding CDR sequence mentioned in Table A-1.

However, as will be clear to the skilled person, the (combinations of) CDR sequences, as well as (the combinations of) CDR sequences and framework sequences mentioned in Table A-1 will generally be preferred.

TABLE A-1 CDR's and framework sequences of Nanobodies against five representative ADAM's (ADAM 8, ADAM 9, ADAM 10. ADAM 17 and ADAM TS5) SEQ FR1 SEQ CDR1 SEQ FR2 SEQ CDR2 Clone against ADAM8 28-B12, 126 EVQLVESGGGL 232 LYTSG 338 WYRQAPG 444 SIASGASGGT VQNGGSLRLS KQREWVA TVYEDSVKG CVMSGSSIS 28- 127 EVQLVESGGGL 233 NYIMG 339 WFRQAPGK 445 AINYEGDRTY D1, H1 VQAGGSLRLSC EREFVA SPNSVKG AASGRTFD 28-A6 128 EVQLVESGGGL 234 DYTMY 340 WFRQAPGK 446 AIGGVTDYAD VQAGGSLRLSC ERELVA SVKG AASGRTFS 28-B6 129 EVQLVESGGGL 235 IRVMG 341 WFRRAPGK 447 TVTWRDNITY VQAGASLKLSC DRESVA YVDSVKG AASGRTYD 28-C1 130 EVQLVESGGGL 236 NYVMG 342 WFRQAPGK 448 AMGDTTLYA VQAGGSLRLSC EREFVA DSVEG AASGRTFS 28-C6 131 EVQLVESGGGL 237 IRVMG 343 WFRRAPGK 449 TTTWRDNITY VQAGASLKLSC GRESVA YMDSVKG AASGRTYD 28-D12 132 EVQLVESGGGL 238 FNAMA 344 WHRQAPG 450 AIHISGNTNY VQAGGSLRLSC NQRELVA ADSVKG ATSGSIAS 28-E1 133 EVQLVESGGGL 239 DYTMA 345 WFRQAPGK 451 AIGSVTDYAD VQAGGSLRLSC ERELVA SVKG TASGRTFS 28-E6 134 EVQLVESGGGL 240 IETMA 346 WHRQAPG 452 AIRSDDMTNY VQAGGSLRLSC KQRELVA PDSVKG AASGNIFR 28-F12 135 EVQLVESGGGL 241 FNSMA 347 WYRQSPG 453 RIFRSANIYY VQAGESLRLSC KQRNLVA DDSVKG KASRNDFS 28-G1 136 EVQLVESGGGL 242 IETMA 348 WHRQAPG 454 AIRRDEMTNY VQAGGSLRLSC KQRELVA LDFVKG AASGNIFR 28-G12 137 EVQLVESGGGL 243 INTMG 349 WYRQTPG 455 SISSGGTTAY VQAGGSLRLSC QQRDWVA ADSVKG AASGSIFS 28-G6 138 EVQLVESGGGL 244 FNAVG 350 WWRQAPG 456 SISTSGSTITP AQAGGSLRLSC TQREWVA YVDSVKG AASGSITS 29-A12 139 EVQLVESGGGL 245 FWMG 351 WFRQTPG 457 AISGSFNVTS VQAGGSLRLSC MKRFFVA YADSVKG AASGRTFS 29-A6 140 FVQLVFSGGGL 246 YDAMA 352 WFRQAPGK 458 VITWSGGTTV VQAGGSLGLS FRFFVA YADSVQG CVFSGRPYS 29-C6 141 FVQLVFSGGFS 247 NFIMG 353 WFRRAPGK 459 AIGDPSTHYA VQVGGSVRLS FRFLLA DSATG CAASGLTFS 29-F12 142 FVQLVFSGGGL 248 RHVIG 354 WFRQAPGK 460 AINSDGDTTT VQAGGSLTLSC FRKFVT DDRSLKG WSGVDFS 29-F6 143 FVQLVFSGGG 249 DYIIG 355 WFRQGPG 461 RISGSGLTNT RVQAGGSLRLS KFRFSVA TTFTVKG CFASGRTFS 29-H1 144 FVQLVFSGGGL 250 IHAMG 356 WYRQAPG 462 SITSSATAYA VQPGGSLRLAC NFRFWVA DSVKG AASKSIDN Clone against ADAM9 41- 145 EVQLVESGGGL 251 TYAMG 357 WFRQAPGK 463 AISWNEVNTY B5, C1, D4 VQAGGSLRLAC EREFVA YTDSVKG 3, E4, E5 AASGRTFS 30-B12, 146 EVQLVESGGGL 252 IYDMG 358 WFRQAPGK 464 VIRWSDGFTY VQAGGSLRLSC EREFVT YEDSVKG AASGPTFS 30-A6 147 EVQLVESGGGL 253 DYAMG 359 WFRQAPEK 465 TINYSGGVTY VQAGGSLRLSC EREFVA YADSVKG AASGRASG 30-B1 148 EVQLVESGGGL 254 RLAMG 360 WFRQAPGK 466 AINWLSESTY VPAGGSLRLSC ERDFVG YEDSVKG AGSGRSFS 30-C1 149 EVQLVESGGGL 255 SYAVG 361 WFRQAPGK 467 VISWTGGSTY VQAGDSLRLSC EREFVA FADSVKG AVSGRTIS 30-D1 150 EVQLVESGGGL 256 SDWMY 362 WVRQAPG 468 SIGIAGTPTFY VQPGGSLRLSC KGPEWVS ADSVKG AASGFAFS 30-D12 151 EVQLVESGGGL 257 DYWG 363 WFRQTPGK 469 RKVWSNGNT VQAGGSLRLSC EREFIG YYIDSVKG ATSGRTFS 30-E12 152 EVQLVESGGGL 258 RLAMG 364 WFRQAPGK 470 AISWLGESTY VPAGGSLRLSC EREFVA YDDSVKG AGSGRTFS 30-G1 153 EVQLVESGGGL 259 NYFMG 365 WFRQAPGK 471 GVAWSSDFT VQAGISLKLSC EREFVA AYTDSVKG TASGGTLT 30-G12 154 EVQLVESGGRL 260 TDVMG 366 WFRQVPEK 472 EIGWRDTTLY VQAGGSLRLSC EREFVA ADSVKG AASGRTFS 31-B1 155 EVQLVESGGGL 261 TLAMG 367 WFRQAPGK 473 AISWSEVNTY VQAGGSLRLAC EREFVA YTDSVKG AASGRTFS 31-D12 156 EVQLVESGGGL 262 NYHVG 368 WFRQAPGK 474 AISASGTSTA VQAGDSLRLSC EREFVA YAGSVKG APSGGIIS 31-E12 157 EVQLVESGGGL 263 AYNMG 369 WFRLRPGK 475 AINWSGGTQ VQAGGSLRLSC EREFVA DYVDSVKG SSSGRTYS 31-F6 158 EVQLVESGGGL 264 TLAMG 370 WFRQAPGK 476 AISWSEVNTY VQAGGSLRLAC EREFVA YTDSVKG AASGRTFS 31-G12 159 EVQLVESGGGL 265 NYVMG 371 WFRQAPGK 477 AITWSDSRTD VQAGESLRLSC ERELVA YADSVKG AASGRTFG 41-A1 160 KVQLVESGGGL 266 TLAMG 372 WFRQAPGK 478 AISWSEVNTY VQAGGSLKLAC FRFFVA YTDSVKG AASGRTFS 41-A3 161 FVQLVFSGGGL 267 TYAMG 373 WFRQAPGK 479 AISRNFVNTY VQAGGSLRLAC FRFFVA YTDSVKG AASGRTFS 41-B1 162 FVQLVFSGGGL 268 GYHMG 374 WFRQAPGK 480 YISSGGAYSN VQAGGSLRLSC FRFFVA YADSVKG TGSDRTFI 41-C5 163 FVQLVFSGGGL 269 AVFMG 375 WYRQLPGK 481 SINRGGSTNY VQAGGSLRLSC QRFLVA APSAKG WSGTITS 41-D1 164 FVQLVFSGGGL 270 TLAMG 376 WFRRAPGK 482 AISWSFDNTY VQAGGSLRLAC FRFFVA YSDSVKG AASGRTFS 41-F6 165 FVQLVFSGGGL 271 TLAMG 377 WFRQAPGK 483 AISWSFVNTY VQAGGSLRLAC FRFFVA YTDSVKG AASGRTFS 30-B6 974 EVHLVESGGG 984 EYALV 994 WFRQAPGK 1004 VAGFAANGIN WQAGGSLRLS EKEREF TDYTDSVKG CVASGLPFS 30-C6 975 EVQLVESGGGL 985 SNTMA 995 WYRQAPRK 1005 SIMTDGTITY VQAGGSLRLSC QREFVA ADSVKG AASGSIFS x30-D6 976 EVQLVESGGGL 986 RLAMG 996 RWFRQAPGK 1006 AISWLAETTY VQPGGSLRLSC EREFVA YEDSVKG AGSGRTFS 30-F12 977 EVQLVESGGGL 987 SYAIA 997 WFRQAPGK 1007 CISSSDGTTY VQPGGSLRLSC EREGVS YADSVKG AASGFTLD Clones against ADAM10 39-A8, 166 EVQLVESGGGL 272 SYCVG 378 WWRQAPG 484 AITRGSNSTD D7, E7, VQTGGSLRLSC KERDWA YVDSVKG F9, G8, AASGRTFT H8, A9 32- 167 EVQLVESGGGL 273 DYAIG 379 WFRQAPGK 485 CISISDSSTYY E12, G12 VQAGGSLRLSC EREGVS ADSVKG AASGFTFG 32- 168 EVQLVESGGGL 274 TYFMG 380 WFRQAPGK 486 FISGNGGSTD E1, E6, VQAGGSLRLSC EREFVA YADSVKG AASERIFS 32-D1 169 EVQLVESGGGL 275 DYAIG 381 WFRQAPGK 487 CISVSDGSTY VQAGGSLRLSC EREGVS YADSVKG AASGFTFD >39- 170 EVQLVESGGGL 276 RYAMG 382 WFRQAPGK 488 AISRSGGNTY D8, E8 VRARGSLRLSC EREFVA FTESVK AASGGTFS 32-06 171 KVQLVESGGGL 277 DYAIG 383 WFRQAPGK 489 CISISDSSTYY VQAGGSLRLSC EREGVS ADSVKG AASGFTFG 32-F1 172 EVQLVESGGGL 278 SYRLG 384 WFRQAPGN 490 SITWSSANTY VQAGGSLTLSC EREFVA YADSVKG AASGRTFS 32-G6 173 EVQLVESGGGL 279 DYAIG 385 WFRQAPGK 491 CISSSDGRTY VQPGGSLRLSC EREGVS YDDSVKG AASGFTFD 33-A1 174 EVQLVESGGGL 280 RYSMG 386 WFRQAPGK 492 YITRSGRTTY VQAGGSLRLSC EREFW YQDSVKG AASGDTFS 33-C6 175 EVQLVESGGGL 281 SYYMG 387 WFRQAPGK 493 HISLSGGNTE VQAGDSLRLSC EREFVA YADSVKG AASGRTFS 33-D1 176 EVQLVESGGGL 282 SDAMG 388 WFRQAPGK 494 GINYNSVYRY VQAGDSLRLSC ERAFVA YTDSVEG AATGRTFS 33-G6 177 EVQLVESGGG 283 SYCVG 389 WWRQAPG 495 AITRGGDTTD SVQTGGSLRLS KERAWA YVDSVKG CAASGRTFT 39-B1 178 EVQLVESGGGL 284 INIMA 390 WYRQAPG 496 AIIGDSTNYA VQAGGSLRLSC KTRDMVA DSVKG ARSGRISN 39-B2 179 EVQLVESGGGL 285 DYAIG 391 WFRQAPGK 497 CISSSDGRTY VQAGGSLRLSC EREGVS YADSVKG AASGFTFD 39-C1 180 EVQLVESGGGL 286 SYVMG 392 WFRQAPGK 498 AVSRSGRNIN VQAGGSLRLSC EREEVA YADLMKG SASGRTFG 39-C2 181 KVQLVESGGGL 287 RYYMG 393 WFRQAPGK 499 FISGTGGSID VQAGGSLRLSC EREEVA YADSVKG AASGRTFS 39-E1 182 EVQLVKSGGGL 288 NNAVG 394 WYRQAPG 500 AMLSGGSTN VQAGGSLRLSC KQREMVA YADSVKG AASGNIFI 39-E2 183 EVQLVESGGGL 289 INIMS 395 WYRQAPG 501 AIIGDATNYA VQAGGSLRLSC KTRDMVA DSVKG GRSGRISN 39-F1 184 EVQLVESGGGL 290 TYFMG 396 WFRQAPGK 502 FISGNGGSTD VQAGGSLRLSC EREFVA YADSVKG AASERIFS 39-G1 185 EVQLVESGGGL 291 INIMS 397 WYRQAPG 503 AIIGDSTNYA VQAGGSLRLSC KTRDMVA DSVKG ARSGRISN 39-G2 186 EVQLVESGGGL 292 DYAIG 398 WFRQAPGK 504 CISMSDGSTY VQAGGSLRLSC EREGVS YADSVKG AASGFTFD 39-C8 187 EMQLVESGGG 293 SYCVG 399 WWRQAPG 505 AITRGSNSTD LVQTGGSLRLS KERDWA YVDSVKG CAASGRTFT 39-D9 188 EVQLVESGGGL 294 SYCVG 400 WWRQAPG 506 AITRGSNSTD VQPGGSLRLAC KERDVVA YVDSVKG AASGRTFT 39-01 189 KVQLVESGGGL 295 NNAVG 401 WYRQAPG 507 AMLSGGSTN VQAGGSLRLSC KQREMVA YADSVKG AASGNIFI Clones against ADAM17 35- 190 EVQLVFSGGGL 296 TYAMG 402 WFRQAPGK 508 GITRSAVSTS B12, E1, VQTGGSLRLSC ERFFVA YTDSVKG G1, F6 AASGGTFS 35-C6, 191 KVQLVFSGGGL 297 TYAMG 403 WFRQAPGK 509 GITRSAVSTS VQTGGSLRLSC FRFFVA YTDSVKG AASGGTFS 40- 192 EVQLVKSGGGL 298 TYAMG 404 WFRQAPGK 510 GITRSGLSTS A2, H2 VQTGGSLRLSC EREFVA YADSVKG IVSGGTFS 40- 193 EVQLVESGGGL 299 TYAMG 405 WFRQAPGK 511 GITRSGLSTS B1, D1 VQTGGSLRLSC EREFVA YADSVKG IVSGGTFS 35- 194 EVQLVKSGGGL 300 TYAMG 406 WFRQAPGK 512 GITRSGVSTS B1, G12 VQTGGSLRLSC FRFFVA YADSVKG AASGGTFS 35-C12 195 KVQLVESGGGL 301 TYAMG 407 WFRQAPGK 513 GITRSAVSTS VQTGGSLRLSC EREFVA YTDSVKG AASGGTFS 35-F12 196 EVQLVESGGGL 302 TYAMG 408 WFRQAPGK 514 GITRSGVSTS VQTGGSLRLSC EREEVA YADSVKG AASGGTFS 35-G6 197 EVQLVESGGGL 303 YAIA 409 WFRQTPGK 515 VINRSGSYVY VQAGDSLRLSC EREFVA YTDSVKG TASGAFGS 40-A1 198 EVQLVESGGGL 304 TYAMG 410 WFRQAPGK 516 GITRSAVSTS VQAGGSLRLSC EREEVA YTDSVKG AASGGTFS 40-B2 199 EVQLVESGGGL 305 TYAMG 411 WFRQAPGK 517 GITRSALSTS VQTGGSLRLSC FREEVA YADSVKG IASGGTFS 40-C1 200 FVQLVFSGGGL 306 YAIA 412 WFRQTPGK 518 VINRSGSYVY VQAGDSLRLSC FREFVA YTDAVKG TASGTFGS 40-D2 201 FVQLVFSGGGL 307 KLAVA 413 WFRQAPGK 519 GITRSAVSTS VQPGGSLRLSC KLAVA YTDSVKG RASGGTFR 40-F1 202 FVQLVFSRGGL 308 TYAMA 414 WFRQAPGK 520 GITRSAVSTS VQTGGSLRLSC EREFVA YADSVKG AASGGTFS 40-F2 203 FVQLVFSGGGL 309 TYAMG 415 WFRQAPGK 521 GITRSSVSTS VQTGGSLRLSC FREFVA YADSVKG AASGGTFS 40-G1 204 EVQLVESGGGL 310 YAIA 416 WFRQTPGK 522 VINRSGSYMY VQAGDSLRLSC EREFVA YIDSVKG TASGTFGS 40-G2 205 EVQQVESGGG 311 TYAMG 417 WFRQAPGK 523 GITRSGVSTS LVQTGGSLRLS EREFVA YADSVKG CIVSGGTFS 40-H1 206 EVQLMESGGG 312 TYAMG 418 WFRQAPGK 524 GITRSAVSTS LVQTGGSLRLS EREFVA YTDSVKG CAASGGTFS 34-A12 978 EVQLVESGGGL 988 YYAIG 998 WFRQAPGK 1018 CISSSDGIRY VQPGGSLRLSC EREGVS YIDSVKG AASGFPLD 34-H1 979 EVQLVESGGGL 989 SYVMG 999 WFRQAPGK 1019 TISRSGESTY VQDGGSLSCA EREFVA YTGSVKG ASGRTFS 35-A12 980 EVQLVESGGGL 990 DYAIG 1000 WFRQAPGK 1020 CIGDKGDRIF VQAGGSLRLSC EREGVS YADSVKG VASGLSFD 35-D1 981 EVQLVESGGGL 991 TYAMA 1001 WFRRAPGK 1021 AIRWSGDRT VQAGGSLRLSC EREFW YYADSVKG AASGRTFS 35-H1 982 EVQLVESGGG 992 SYAMG 1002 WFRQAPGK 1022 GINGSGNGIR WVQAGDSLRL EREFVA IALSVRG SCAASGRTFS Clone against ADAM TS5 36-A6, 207 EVQLVESGGGL 313 SYNMG 419 WFRQAPGK 525 ADMWSGTTT 40-H8 VQAGDSLRLSC EQEFVA YYTDSVKG AASGRTLS 36-A1 208 EVQLVESGGGL 314 IRAMG 420 WLRQAPGN 526 AISRDGDRTY VQPGGSLTLSC EREFVA YTDWKG AASGGTFS 36-06 209 EVQLVESGGGL 315 SRYHMG 421 WFRQGPG 527 AVSSLGPFTR VQAGGSLRLSC KEREFVA YADSVKG AFSDGRTV 36-D6 210 EVQLVESGGGL 316 NYAMA 422 WFRQAPGK 528 GINWSGNGV VQAGGSLRLSC EREFVA YYPDSLKE VASGLTFR 36-E1 211 EVQLVESGGGL 317 SYNMG 423 WFRQAPGK 529 AIMWSGTTTY VQAGDSLRLSC ELEFVA YTDSVKG AASGRTLS 36-F1 212 EVQLVESGGGL 318 IRAMG 424 WFRQAPGN 530 AISRDGDRTY VQPGGSLTLSC EREFVA YTDWKG AASGGTFS 37-B1 213 EVQLVESGGGL 319 PYKVA 425 WFRQAPGK 531 VIHWYGITAY VQPGQSLRLSC ERDFVA ADTVKG AASGRTLN 37-B12 214 EVQLVESGGGL 320 NYHMA 426 WFRQAPGK 532 DFRPSGGSP VQAGGSLRLSC EQEFVG YYANYADSV AASGRSLR KG 37-B6 215 EVQLVESGGGL 321 IYPIG 427 WFRQAPGK 533 AIDWSGSRS VQAGGSLRLSC EREFVA YYLDSMKG TAAGRTHS 37-C12 216 EVQLVESRGGL 322 GYGVG 428 WFRQGPG 534 AISWSPGRT VQTGGSLRLSC KDREFVA DYGDAVKG TTSGGTLR 37-06 217 EVQLVESGGG 323 NYGVG 429 WFRQGPG 535 AISWSPGRT WQTGGSLRLS KDREFVA DYGDAVKG CTTSGGTFR 37-D6 218 EVQLVESGGGL 324 PYKVA 430 WFRQAPGK 536 VIHWYGITAY VRAGGSLRLSC ERDFVA ADTVKG AASGRTLN 37-E6 219 EVQLVESGGGL 325 TYHMA 431 WFRQAPGK 537 DLRPSGGRA VQAGGSLRLSC EQEFVG GYADYADSV + AASGRSLG KG 37-F1 220 KVQLVESGGGL 326 PYKVA 432 WFRQAPGK 538 VIHWYGITAY VRAGGSLRLSC ERDFVA ADTVKG AASGRTLN 37-F12 221 EVQLVESGGGL 327 NYGVG 433 WFRQGPG 539 AISWSPGRT VQTGGSLRLSC KDREFVA DYGDAVKG TTSGGTFR 37-G1 222 EVQLVESGGGL 328 MGRVG 434 WFRQDPGK 540 AISRSGDTTY VQAGGSLRLTC EREFVA YDDSVKD AASGRTFS 37-G6 223 EVQLVESGGGL 329 PYKVA 435 WFRQAPGK 541 VIHWYGITAY VRAGGSLRLSC ERDFVA ADTVKG AASGRTLN 40-A7 224 EVQLVESGGGL 330 MYTMG 436 WFRQAPGK 542 AITPINWGGR VQAGASLRLSC ERDFVA GTHIADSVKG GASGRTLS 40-B8 225 EVQLVESGGGL 331 FTNYAIA 437 WFRQAPGK 543 WNWSTYYA VQAGGSLRLSC ERKFVA DSVKG AASDSART 40-D7 226 EVQLMESGGG 332 SYTMA 438 WFRQAPGK 544 YVFGGGEITD LVQAGGSLRLS EREEVA YADFVKG CAASGRTIS 40-F7 227 EVQLVESGGGL 333 NYAMA 439 WFRQAPGK 545 GINWSGNGV VQAGGSLRLSC EREEVA YYPDSLKE VASGLTFR 40-F8 228 EVQLVESGGGL 334 MYGSG 440 WFRRAPGK 546 FINHSGGRTN VQAGGSLRLSC EREFVG YADSVKG AASGLTFR 40-G7 229 EVQLVESGGGL 335 NYAMA 441 WFRQAPGK 547 GINWSGNGV VQAGGSLRLSC EREFVA YYPDSLKE VASGLTFR 40-G8 230 EVQLVESGGGL 336 MYTMG 442 WFRQAPGK 548 AITPINWGGR VQAGASLRLSC ERDFVA GTHIADSVKG GASGRTLN 40-H7 231 EVQLVESGGGL 337 SYTMA 443 WFRQAPGK 549 YVFGGGEITD VQAGGSLGLS EREFVA YADFVKG CAASGRTIS x40-E8 983 EVQLVESGGTL 993 AYGTG 1003 WFRQAPGK 1013 TISWSGSTTS VXAGGSLRLSC EREFVA YADSVKG AASGLAFN SEQ FR3 SEQ CDR3 SEQ FR4 Clone against ADAM8 28-B12, 550 RFTISRDDAENT 656 QDPMRTR 762 WGQGTQVTVSS VYLQMITLKPED DAY TAVYYCRA 28- 551 RFTISRDNAKNT 657 GPRYGKY 763 WGQGTQVTVSS VYLQMNSLKPED DY D1, H1 TAVYQCAT 28-A6 552 RFTIARDNAKST 658 KHRSVAT 764 WGQGTQVTVSS VSLQMNSLKPED RTGGNVS TAVYYCAA 28-B6 553 RFTITRDNAKNT 659 QTEDSAQ 765 WGQGTQVTVSS VYLQMNNLKPED YIY TAVYYCAA 28-C1 554 RFTISRDNDQNT 660 RSRFIPP 766 WGQGTQVTVSS VYLQMSSLKPED LTFRTGG TAAYFCAA AYDY 28-C6 555 RFTITRDNAKNT 661 QTEDSAQ 767 WGQGTQVTVSS VYLQMNNLKPED YIY TAVYYCAA 28-D12 556 RFTISRDNGKNT 662 IEVRTGL 768 WGQGTQVTVSS VYLQMNSLRPED SPRGH TAVYYCNA 28-E1 557 RFTISRDNAKNT 663 KHRTRTR 769 WGQGTQVTVSS VSLQMNSLKPED GYVN TAVYYCAA 28-E6 558 RFTISRDNFKNT 664 IQRRAPY 770 WGQGTQVTVSS VYLQMNSLTPED SRLETY TAVYYCNL 28-F12 559 RFTISRDSALNTV 665 RLSNGLDY 771 WGQGTQVTVSS FLQMNALKSEDT AVYYCNG 28-G1 560 RFTISRDNFKNT 666 IQRRAPY 772 WGQGTQVTVSS VYLQMNSLKPED SRLETY TAVYYCNL 28-G12 561 RFSISRDGPKNT 667 QRRWSQDY 773 WGQGTQVTVSS VYLQMNSLKPED TAVYYCKA 28-G6 562 RFTISKDNTKNT 668 TERMHHSY 774 CGQGTQVTV VYLQMNSLKPED TAVYYCCA 29-A12 563 RFTISRDNAKNT 669 QRWRGGSY 775 WGQGTQVTVSS VTLQMNSLKPFD FY TAVYYCAA 29-A6 564 RFTISRDIAKNTM 670 SAGARYGV 776 WGQGTQVTVSS NLQMNSLKPDDT GWWRNGQNY AVYYCAG QN 29-C6 565 RFTISRDNAKNM 671 RSRYSTGT 777 WGQGTQVTVSS VYLQMNSLKTFD LYDQTKYI TAVYYCAA Y 29-F12 566 RFTISRFNANNT 672 GPQYRSYF 778 WGQGTQVTVSS VHLQMTNLKVFD ARSYLY TAIYYCAT 29-F6 567 RFTISRDNAKNT 673 AYSGHFSGR 779 WGQGTQVTVSS VYLQMNSLKPFD VSDFLY TAVYYCAA 29-H1 568 RFIISRDNAENTI 674 QVLVPGGR 780 WGQGTQVTVSS YLQMHSLKPFDT NDY AVYYCKG Clone against ADAM9 41- 569 RFTISRNNAENT 675 DRHYTAQQ 781 WGKGTLVTVSS B5, C1, D4 VYLQMNSLKRED MRVMTGAS 3, E4, E5 TAVYYCAS YMDY 30-B12, 570 RFTISRDNAKNT 676 NTAPLRII 782 WGQGTQVTVSS VYLQMNNLKPED NFRNAYNY TAVYVCAA DY 30-A6 571 RFTISRDNAKNT 677 DRHYGAYA 783 RGQGTQVTVSS VYLQMNSLKPED LLGLGTHA TAVYYCAA YIDY 30-B1 572 RFTISRDNAKNT 678 DRFATAQN 784 WGQGTQVTVSS VYLQMNSLKPED MNTMGSLD TAVYYCAS Y 30-C1 573 RFTISRDNAKNT 679 DRFTTGSG 785 WGQGIQVTVSS VYLQMNNLKSED RTSYPRPS TAVYYCTA EFDH 30-D1 574 RFTISRDNANNM 680 EGIYCSNW 786 WGQGTQVTVSS LYLQMTSLKPGD RCLFGPKT TALYYCAR DLPAS 30-D12 575 RFTISGDNAKRT 681 RSPMSPTW 787 WGQGTQVTVSS VYLQMNSLKPED DN TALYYCAA 30-E12 576 RFTISRDNAKNT 682 DRFATAQA 788 WGQGTQVTVSS VYLQMNSLKPED MGAMGSLD TAVYYCAS Y 30-G1 577 RFTISRDNAKNT 683 RRRGGGYN 789 WGQGTQVTVSS VYLQMNDVKPE QLNLYDY DTAVWYCAA 30-G12 578 RFTISRDNAKNM 684 RRAAAAYDQ 790 WGQGTQVTVSS VYLQMNSLKPED TAVYYCSS 31-B1 579 RFTISRNNAENT 685 DRHYSAQQ 791 WKGKTLVTVSS VYLQMNSLKPED MRVMTGAS TAVYYCAA YMDY 31-D12 580 RFTISRDNAKNT 686 SEYVFRYY 792 WGQGTQVTVSS AFLQMNTLKPED DDSRYYAY TAVYYCAA 31-E12 581 RFTAIADVAKKTV 687 TQWGSSGW 793 WGQGTQVTVSS FLQMTSLKPEDT KQARWYDF AVYYCAA 31-F6 582 RFTISRNNAENT 688 DRHYTAQQ 794 WGKGTLVTVSS VYLQMNSLKRED MRVMTGAS TAVYYCAS YMDY 31-G12 583 RFTISRDNVKNL 689 SSIGPYRL 795 WGRGTQVTVSS VYLQMNSLRPED LDSSRYAY TAVYSCAA 41-A1 584 RFTISRNNAENT 690 DRHYSAQQ 796 WGKGTLVTVSS VYLQMNSLKPED MRVMTGAS TAVYYCAA YMDY 41-A3 585 RFTISRNNAENT 691 DRHYTAQQ 797 WGKGTLVTVSS VYLQMNSLKRFD MRVMTGAS TAVYYCAS YMDY 41-B1 586 RFTISRDNAKNT 692 TDYNKAYA 798 WGQGTQVTVSS AYLQMNSLKPFD RFGRRYDY TAVYHCAA 41-C5 587 RFTISRVNANST 693 VINTRSF 799 WGQGTQVTVSS MYLQMNSLQPF DTAVYYCYG 41-D1 588 RFTISRNNAEST 694 DRHLLAQQMR 800 WGKGTLVTVSS VYLQMNSLKPFD VMTGASYMDY TAVYYCAS 41-F6 589 RFTISRNNAENT 695 DRHATAQHMR 801 WGKGTLVTVSS VYLQMNSLKPFD VMTGASYMDY  TAVYYCVS 30-B6 1014 RFTISRDNAKNT 1024 SWTPAYNDIPR 1034 WGKGTLVTVSS VYLQMNSLKPED HMSSMDQ TAVYYCAA 30-C6 1015 RFAISRDTAKNT 1025 RQYGEYWQAA 1035 WGQGTQVTVSS VALQMNSLKPED GS TAVYYCNA x30-D6 1016 RFTISRDNAKNT 1026 DRFATAQHMG 1036 WGQGTQVTVSS VYLQMNSLKPED AMGSLDY TAVYYCAS 30-F12 1017 RFTISRDNAKNM 1027 DPELVTVCRPG 1037 WGQGTQVTVSS VYLQMQSLKPED WGPAYDY TAVYYCAA Clones against ADAM10 39-A8, D7, 590 RFTISRDNAENT 696 DINCRNLYTGR 802 WGQGTQVTVSS E7, F9, G8, VYLQMNSLKPED PEY H8, A9 TAVYYCAA 32-E12, G12 591 RFTISSDNAKNT 697 DRLAYGLDPNF 803 WGQGTQVTVSS VYLQMNSLKPED YDY TAVYYCAA 32-E1, E6, 592 RFAISRDNVKNT 698 AGRQIKSTWD 804 WGQGTQVTVSS LYLQMSSLKPDD Y TAVYYCAV 32-D1, 593 RFTISSDNAKNT 699 DRLAYGLDPNF 805 WGQGTQVTVSS VYLQMNSLKPED YDH TAVYYCAA >39-D8, E8 594 GRFTMSRDNAK 700 RSAAVYTTTVY 806 WGQGTQVTVSS NTVYLQMNSLKP LVDFEYNY EDTAVYYCAA 32-C6 595 RFTISSDNAKNT 701 DRLAYGLDPNF 807 WGQGTQVTVSS VYLQMNSLKPED YDY TAVYYCAA 32-F1 596 RFTISRERAKNT 702 EDVGKPFDS 808 WGQGTQVTVSS MYLQMDSLRPE DTAVYYCAK 32-G6 597 RFTISSDNAKNT 703 DRLAYGLDPNF 809 WGQGTQVTVSS VYLQMNSLKPED YDY TAVYYCAA 33-A1 598 RFTISRDNAKNT 704 MRSGNVRYPE 810 WGQGTQVTVSS VYLQMNSLKPED RYDY TAVYYCAA 33-C6 599 RFTITRDNAGNT 705 SPSLRSAWQY 811 WGQGTQVTVSS VYLQMNSLKPED TGVYCCAA 33-D1 600 RFTISRDNAKNT 706 DSDYYSLIGGR 812 WGQGTQVTVSS VYLQMNSLKPED PVNY TAVYYCAA 33-G6 601 RFTISRDKAENT 707 DINCRNLYTGR 813 WGRGTQVTVSS VYLQMNSLKPED PEY TAVYYCAA 39-B1 602 RFTISRDNAKNT 708 SGVD 814 WGQGTQVTVSS VYLHMNRLKPED TGVYYCKI 39-B2 603 RFTISSDNAKNT 709 DRLAYGLDPNF 815 WGQGTQVTVSS VYLQMNSLKPED YDY TAVYYCAA 39-C1 604 RFTVSRDNTKNT 710 DGTISSSWADL 816 WGQGTQVTVSS VYLQLNNLTPED RRGETYGD TAVYYCAA 39-C2 605 RFTISRDSAKNT 711 ASGNSGRSTW 817 WGQGTQVTVSS VYLQMNSLKPED DY TAVYYCAA 39-E1 606 RFTISRDNAKNT 712 QVNGIWAR 818 WGQGTQVTVSS VYLQMNSLKPED TAVYYCNV 39-E2 607 RFTISRDNAKNA 713 PGVD 819 WGQGTQVTVSS VSLHMNRLKPED TGVYYCKI 39-F1 608 RFAISRDNVKNT 714 AGRQIKSTWG 820 WGQGTQVTVSS LYLQMSSLKPDD Y TAVYYCAV 39-G1 609 RFTISRDNAKNT 715 PGVD 821 WGQGTQVTVSS VHLQMNRLKPE DTGVYYCNI 39-G2 610 RFTISSDNAKNT 716 DRLAFGLDSNF 822 WGQGTQVTVSS VYLQMNSLKPED YDY TAVYYCAA 39-C8 611 RFTISRDNAENT 717 DINCRNLYTGR 823 WGQGTQVTVSS VYLQMNSLKPED PEY TAVYYCAA 39-D9 612 RFTISRDNAENT 718 DINCRNLYTGR 824 WGQGTQVTVSS VYLQMNSLKPED PEY TAVYYCAA 39-d1 613 RFTISRDNAKNT 719 QVNGTNAR 825 WGQGTQVTVSS VYLQMNSLKPED TAVYYCNV Clones against ADAM17 35- 614 RFTISRDNAKDM 720 TRRLHFGNGA 826 WGKGTPVTVSS B12, E1, VYLQMNSLKPED DY G1, F6 TALYYCAA 35-C6,  615 RFTISRDNAKDM 721 TRRLHFGNGA 827 WGKGTPVTVSS VYLQMNSLKPFD DY TALYYCAA 40-A2, H2 616 RFTISRDNAKDT 722 TRRLHFGNGA 828 WGKGTPVTVSS VYLQMNSLKPED DY TALYYCAA B1, D1 617 RFTISRDNAKDT 723 TRRLHFGNGA 829 WGKGTPVTVSS VYLQMNSLKPED DY TALYYCAA 35-B1, G12 618 RFTISRDNAKDM 724 TRRLHFGNGA 830 WGKGTPVTVSS VYLQMNSLKPFD DY TALYYCAA 35-C12 619 RFTISRDNAKDM 725 TRRLHFGNGA 831 WGKGTPVTVSS VYLQMNSLKPED DY TALYYCAT 35-F12 620 RFTISRDNAKDM 726 TRRLHFGNGA 832 WGKGTPVTVSS VYLQMNSLKPED DY TALYSCAA 35-G6 621 RFTISRHNAKNT 727 RDGTLYSTTYY 833 WGQGTQVTVSS AYLQMNSLKPED YISSYTY TAVYYCAA 40-A1 622 RFTISRDNAKDM 728 TRRLHFGNGA 834 WGKGTPVTVSS VYLQMNSLKPED DY TALYYCAA 40-B2 623 RFTISRDNAKDM 729 TRRLHFGNGA 835 WGKGTPVTVSS VYLQMNSLKPED DY TALYYCAA 40-C1 624 RFTISRHNAKNT 730 RDGTLYSTTYY 836 WGQGTQVTVSS AYLQMNSLKPED YISSYTY TAVYYCAA 40-D2 625 RFTISRDNAKDM 731 TRRLHFGNGA 837 WGKGTPVTVSS VYLQMNSLKPED DY TALYYCAA 40-F1 626 RFTISRDNAKDM 732 TRRLHFGNGA 838 WGKGTPVTVSS VYLQMNSLKPED DY TALYYCAA 40-F2 627 RFTISRDNAKDM 733 TRRLHFGNGA 839 WGKGTPVTVSS VYLQMNSLKPFD  DY TALYYCAA 40-G1 628 RFTISRHNAKNT 734 RDGTLYSTSYY 840 WGQGTQVTVSS AYLQMNSLKPED YISSYTY TAVYYCAA 40-G2 629 RFTISRDNAKDT 735 TRRLHFGNGA 841 WGKGTPVTVSS VYLQMNSLKPED DY TALYYCAA 40-H1 630 RFTISRDNAKDM 736 TRRLHFGNGA 842 WGKGTPVTVSS VYLQMNSLKPED DY TALYYCAA 34-A12 1028 RFTISRDNAKNM 1028 DPIRICSSQPR 1038 WGQGTQVTVSS VYLQMNSLKPED RYDY TAVYYCAA 34-H1 1029 RFTISRDNAENT 1029 DYNPYQSGSY 1039 WGQGTQVTVSS VYLQMNSLKPED YSSRSSTYDY TAVYYCAA 35-A12 1030 RFAISSDHAKHT 1030 VAPFAFCTESL 1040 WGQGIQVTVSS VDLQMTNLKPED PDTWYDY TATYYCAA 35-D1 1031 RFTISRDNAKNT 1031 RRFSGFDYSG 1041 WGQGTQVTVSS VYLQMNSLKPED NYYAWDAYDY TAVYYCAL 35-H1 1032 RFTITRDNAKNT 1032 TFAAGASEYHY 1042 WGQGTQVTVSS GYLQMNSLKRE DTAVYYCGP Clone against ADAM TS5 36-A6, 631 RFTISRDNAKNM 737 ELHSSDYTSPG 843 WGQGTQVTVSS 40-H8 VYLQMNSLKPED AYAY TAVYYCAA 36-A1 632 RFTISRDNAKST 738 TRPFKVLTATI 844 WGQGTQVTVSS VYLQINSLKTEDT ENDFTY AVYYCAA 36-C6 633 RFTISRDNAKNA 739 DSSGYSGSYS 845 WGQGTQVTVSS VYLQMNSLKPED SEYRYDY TAVYYCAA 36-D6 634 RFAISRENPKNM 740 DRTLTAWDRD 846 WGQGTQVTVSS VYLQMNSLNPED NAEY TAVYYCTA 36-E1 635 RFTISTDNAKNM 741 ELHSSDYTSPG 847 WGQGTQVTVSS VYLQMNSLKPED AYAY TAVYYCAA 36-F1 636 RFTISRDNAKST 742 TRPFKVLSAIIE 848 WGQGTQVTVSS VYLQINSLKTEDT NDFTY AVYYCAA 37-B1 637 RFSISRDNAKGT 743 DSTSALGHATT 849 WGQGTQVTVSS VYLQMDSLKPED DFDS TAVYYCAL 37-B12 638 RFTIFRDNAKNT 744 DSHGGIAFMEP 850 WGQGTQVTVSS VYLQMNSLKLED DEYDY TAVYYCAA 37-B6 639 RFTISGDNAKNT 745 RPVGDFADPR 851 WGQGTQVTVSS VYLQMNSLKPED YRF TGVYYCAA 37-C12 640 RFTISRDNAKNT 746 DSVSASYYDA 852 WGRGTQVTVSS VYLQMNSLKPED RNDMAYDY TAVYYCAA 37-C6 641 RFTISRDNPKNT 747 DSVSASYYDA 853 WGRGTQVTVSS VYLQMNSLKPED RNDMAYDY TAVYYCAA 37-D6 642 RFSISRDNAQGT 748 DSTSALGHTTS 854 WGQGTQVTVSS VKLQMDSLKPED DFDS TAVYYCAL 37-E6 643 RFTIFRDNAKNT 749 DSHGGISFMEP 855 WGQGTQVTVSS VYLQMNSLKLED DEYDY TAVYYCAA 37-F1 644 RFSISRDNAKGT 750 DSTSALGHATT 856 WGQGTQVTVSS VYLQMDSLKPED DFDS TAVYYCAL 37-F12 645 RFTISRDNAKNT 751 DSVSASYYDA 857 WGRGTQVTVSS VYLQMNSLKPED RNDMAYDY TAVYYCAA 37-G1 646 RFTTSRDNAKNT 752 ATFRAVRQDP 858 WGQGTQVTVSS IDLRMNSLKPED SYSASYDY TAVYYCAA 37-G6 647 RFSISRDNAKGT 753 DSTSALGHATT 859 WGQGTQVTVSS VYLHMDSLKPED DFDS TAVYYCAL 40-A7 648 RFTIFRDNTKNTI 754 ESHGSTSPRN 860 WGQGTQVTVSS NLQMNNLNPED PLQYDY TAVYYCAA 40-B8 649 RFTISRDNAKNT 755 GDYRSDYRSP 861 WGQGTQVTVSS VNLQMNSLKPED VAYNY TAVYYCAV 40-D7 650 RFTISKDNAKNT 756 ECVGDVYRSR 862 WGQGTQVTVSS GYLQMNSLKPE DYTY DTAVYYCAM 40-F7 651 RFAISRENAKNM 757 DRTLTAWDRD 863 WGQGTQVTVSS VYLQMNSLNPED SAEY TAVYYCTA 40-F8 652 RFTISRDNAKDT 758 PISGYINPAVY 864 WGQGTQVTVSS VYLQMNSLKPED DRPGSYDY TAVYYCAV 40-G7 653 RFAISRENSKNM 759 DRTLTAWDRE 865 WGQGTQVTVSS VYLQMNSLNPED SAEY TAVYYCTA 40-G8 654 RFTIFRDNTKNTI 760 ESHGSTSPRN 866 WGQGTQVTVSS ILQMNNLNPEDTA PLQYDY VYYCAA 40-H7 655 RFTISKDNAKNT 761 ECVGDVYRSR 867 WGQGTQVTVSS GYLQMNSLKPE DYTY DTAVYYCAM x40-E8 1023 RFTVSRDNAKNT 1033 NRYGLVYKEE 1043 WGQGTQVTVSS VYLQMNSLKPED RHYDF TAIYYCAM

Thus, in the Nanobodies of the invention, at least one of the CDR1, CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1; or from the group of CDR1, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% “sequence identity” (as defined herein) with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 “amino acid difference(s)” (as defined herein) with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

In this context, by “suitably chosen” is meant that, as applicable, a CDR1 sequence is chosen from suitable CDR1 sequences (i.e. as defined herein), a CDR2 sequence is chosen from suitable CDR2 sequences (i.e. as defined herein), and a CDR3 sequence is chosen from suitable CDR3 sequence (i.e. as defined herein), respectively. More in particular, the CDR sequences are preferably chosen such that the Nanobodies of the invention bind to a metalloproteinase from the ADAM family with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A) value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein.

In particular, in the Nanobodies of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table A-1 or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table A-1; and/or from the group consisting of the CDR3 sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR3 sequences listed in Table A-1.

Preferably, in the Nanobodies of the invention, at least two of the CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1 or from the group consisting of CDR1, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 “amino acid difference(s)” with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

In particular, in the Nanobodies of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 sequences listed in Table A-1 or from the group of CDR3 sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 sequences listed in Table A-1, respectively; and at least one of the CDR1 and CDR2 sequences present is suitably chosen from the group consisting of the CDR1 and CDR2 sequences, respectively, listed in Table A-1 or from the group of CDR1 and CDR2 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR1 and CDR2 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1 and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR1 and CDR2 sequences, respectively, listed in Table A-1.

Most preferably, in the Nanobodies of the invention, all three CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1 or from the group of CDR1, CDR2 and CDR3 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, at least one of the CDR1, CDR2 and CDR3 sequences present is suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1. Preferably, in this aspect, at least one or preferably both of the other two CDR sequences present are suitably chosen from CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences, respectively, listed in Table A-1.

In particular, in the Nanobodies of the invention, at least the CDR3 sequence present is suitably chosen from the group consisting of the CDR3 listed in Table A-1. Preferably, in this aspect, at least one and preferably both of the CDR1 and CDR2 sequences present are suitably chosen from the groups of CDR1 and CDR2 sequences, respectively, that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the CDR1 and CDR2 sequences, respectively, listed in Table A-1; and/or from the group consisting of the CDR1 and CDR2 sequences, respectively, that have 3, 2 or only 1 amino acid difference(s) with at least one of the CDR1 and CDR2 sequences, respectively, listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, at least two of the CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1. Preferably, in this aspect, the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table A-1; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with at least one of the corresponding sequences listed in Table A-1.

In particular, in the Nanobodies of the invention, at least the CDR3 sequence is suitably chosen from the group consisting of the CDR3 sequences listed in Table A-1, and either the CDR1 sequence or the CDR2 sequence is suitably chosen from the group consisting of the CDR1 and CDR2 sequences, respectively, listed in Table A-1. Preferably, in this aspect, the remaining CDR sequence present is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the corresponding CDR sequences listed in Table A-1; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with the corresponding CDR sequences listed in Table A-1.

Even more preferably, in the Nanobodies of the invention, all three CDR1, CDR2 and CDR3 sequences present are suitably chosen from the group consisting of the CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

Also, generally, the combinations of CDR's listed in Table A-1 (i.e. those mentioned on the same line in Table A-1) are preferred. Thus, it is generally preferred that, when a CDR in a Nanobody of the invention is a CDR sequence mentioned in Table A-1 or is suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with a CDR sequence listed in Table A-1; and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with a CDR sequence listed in Table A-1, that at least one and preferably both of the other CDR's are suitably chosen from the CDR sequences that belong to the same combination in Table A-1 (i.e. mentioned on the same line in Table A-1) or are suitably chosen from the group of CDR sequences that have at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the CDR sequence(s) belonging to the same combination and/or from the group consisting of CDR sequences that have 3, 2 or only 1 amino acid difference(s) with the CDR sequence(s) belonging to the same combination. The other preferences indicated in the above paragraphs also apply to the combinations of CDR's mentioned in Table A-1.

Thus, by means of non-limiting examples, a Nanobody of the invention can for example comprise a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1, a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table A-1 (but belonging to a different combination), and a CDR3 sequence.

Some preferred Nanobodies of the invention may for example comprise: (1) a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1; a CDR2 sequence that has 3, 2 or 1 amino acid difference with one of the CDR2 sequences mentioned in Table A-1 (but belonging to a different combination); and a CDR3 sequence that has more than 80% sequence identity with one of the CDR3 sequences mentioned in Table A-1 (but belonging to a different combination); or (2) a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1; a CDR2 sequence, and one of the CDR3 sequences listed in Table A-1; or (3) a CDR1 sequence; a CDR2 sequence that has more than 80% sequence identity with one of the CDR2 sequence listed in Table A-1; and a CDR3 sequence that has 3, 2 or 1 amino acid differences with the CDR3 sequence mentioned in Table A-1 that belongs to the same combination as the CDR2 sequence.

Some particularly preferred Nanobodies of the invention may for example comprise: (1) a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1; a CDR2 sequence that has 3, 2 or 1 amino acid difference with the CDR2 sequence mentioned in Table A-1 that belongs to the same combination; and a CDR3 sequence that has more than 80% sequence identity with the CDR3 sequence mentioned in Table A-1 that belongs to the same combination; (2) a CDR1 sequence; a CDR 2 listed in Table A-1 and a CDR3 sequence listed in Table A-1 (in which the CDR2 sequence and CDR3 sequence may belong to different combinations).

Some even more preferred Nanobodies of the invention may for example comprise: (1) a CDR1 sequence that has more than 80% sequence identity with one of the CDR1 sequences mentioned in Table A-1; the CDR2 sequence listed in Table A-1 that belongs to the same combination; and a CDR3 sequence mentioned in Table A-1 that belongs to a different combination; or (2) a CDR1 sequence mentioned in Table A-1; a CDR2 sequence that has 3, 2 or 1 amino acid differences with the CDR2 sequence mentioned in Table A-1 that belongs to the same combination; and a CDR3 sequence that has more than 80% sequence identity with the CDR3 sequence listed in Table A-1 that belongs to the same or a different combination.

Particularly preferred Nanobodies of the invention may for example comprise a CDR1 sequence mentioned in Table A-1, a CDR2 sequence that has more than 80% sequence identity with the CDR2 sequence mentioned in Table A-1 that belongs to the same combination; and the CDR3 sequence mentioned in Table A-1 that belongs to the same combination.

In the most preferred Nanobodies of the invention, the CDR1, CDR2 and CDR3 sequences present are suitably chosen from one of the combinations of CDR1, CDR2 and CDR3 sequences, respectively, listed in Table A-1.

According to another preferred, but non-limiting aspect of the invention (a) CDR1 has a length of between 1 and 12 amino acid residues, and usually between 2 and 9 amino acid residues, such as 5, 6 or 7 amino acid residues; and/or (b) CDR2 has a length of between 13 and 24 amino acid residues, and usually between 15 and 21 amino acid residues, such as 16 and 17 amino acid residues; and/or (c) CDR3 has a length of between 2 and 35 amino acid residues, and usually between 3 and 30 amino acid residues, such as between 6 and 23 amino acid residues.

In another preferred, but non-limiting aspect, the invention relates to a Nanobody in which the CDR sequences (as defined herein) have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053.

Generally, Nanobodies with the above CDR sequences may be as further described herein, and preferably have framework sequences that are also as further described herein. Thus, for example and as mentioned herein, such Nanobodies may be naturally occurring

Nanobodies (from any suitable species), naturally occurring V_(HH) sequences (i.e. from a suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences or Nanobodies, including but not limited to partially humanized Nanobodies or V_(HH) sequences, fully humanized Nanobodies or V_(HH) sequences, camelized heavy chain variable domain sequences, as well as Nanobodies that have been obtained by the techniques mentioned herein.

Thus, in one specific, but non-limiting aspect, the invention relates to a humanized Nanobody, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which CDR1 to CDR3 are as defined herein and in which said humanized Nanobody comprises at least one humanizing substitution (as defined herein), and in particular at least one humanizing substitution in at least one of its framework sequences (as defined herein).

In another preferred, but non-limiting aspect, the invention relates to a Nanobody in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said Nanobody and one or more of the sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053, in which the amino acid residues that form the framework regions are disregarded. Such Nanobodies can be as further described herein.

In another preferred, but non-limiting aspect, the invention relates to a Nanobody with an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 868 to 973 and/or 1044 to 1053 or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053.

Another preferred, but non-limiting aspect of the invention relates to humanized variants of the Nanobodies of SEQ ID NO's: 868 to 973 and/or 1044 to 1053, that comprise, compared to the corresponding native V_(HH) sequence, at least one humanizing substitution (as defined herein), and in particular at least one humanizing substitution in at least one of its framework sequences (as defined herein).

The polypeptides of the invention comprise or essentially consist of at least one Nanobody of the invention.

It will be clear to the skilled person that the Nanobodies that are mentioned herein as “preferred” (or “more preferred”, “even more preferred”, etc.) are also preferred (or more preferred, or even more preferred, etc.) for use in the polypeptides described herein. Thus, polypeptides that comprise or essentially consist of one or more “preferred” Nanobodies of the invention will generally be preferred, and polypeptides that comprise or essentially consist of one or more “more preferred” Nanobodies of the invention will generally be more preferred, etc.

Generally, proteins or polypeptides that comprise or essentially consist of a single Nanobody (such as a single Nanobody of the invention) will be referred to herein as “monovalent” proteins or polypeptides or as “monovalent constructs”. Proteins and polypeptides that comprise or essentially consist of two or more Nanobodies (such as at least two Nanobodies of the invention or at least one Nanobody of the invention and at least one other Nanobody) will be referred to herein as “multivalent” proteins or polypeptides or as “multivalent constructs”, and these may provide certain advantages compared to the corresponding monovalent Nanobodies of the invention. Some non-limiting examples of such multivalent constructs will become clear from the further description herein.

According to one specific, but non-limiting aspect, a polypeptide of the invention comprises or essentially consists of at least two Nanobodies of the invention, such as two or three Nanobodies of the invention. As further described herein, such multivalent constructs can provide certain advantages compared to a protein or polypeptide comprising or essentially consisting of a single Nanobody of the invention, such as a much improved avidity for a metalloproteinase from the ADAM family. Such multivalent constructs will be clear to the skilled person based on the disclosure herein.

According to another specific, but non-limiting aspect, a polypeptide of the invention comprises or essentially consists of at least one Nanobody of the invention and at least one other binding unit (i.e. directed against another epitope, antigen, target, protein or polypeptide), which is preferably also a Nanobody. Such proteins or polypeptides are also referred to herein as “multispecific” proteins or polypeptides or as ‘multispecific constructs”, and these may provide certain advantages compared to the corresponding monovalent Nanobodies of the invention (as will become clear from the further discussion herein of some preferred, but-nonlimiting multispecific constructs). Such multispecific constructs will be clear to the skilled person based on the disclosure herein.

According to yet another specific, but non-limiting aspect, a polypeptide of the invention comprises or essentially consists of at least one Nanobody of the invention, optionally one or more further Nanobodies, and at least one other amino acid sequence (such as a protein or polypeptide) that confers at least one desired property to the Nanobody of the invention and/or to the resulting fusion protein. Again, such fusion proteins may provide certain advantages compared to the corresponding monovalent Nanobodies of the invention. Some non-limiting examples of such amino acid sequences and of such fusion constructs will become clear from the further description herein.

It is also possible to combine two or more of the above aspects, for example to provide a trivalent bispecific construct comprising two Nanobodies of the invention and one other Nanobody, and optionally one or more other amino acid sequences. Further non-limiting examples of such constructs, as well as some constructs that are particularly preferred within the context of the present invention, will become clear from the further description herein.

In the above constructs, the one or more Nanobodies and/or other amino acid sequences may be directly linked to each other and/or suitably linked to each other via one or more linker sequences. Some suitable but non-limiting examples of such linkers will become clear from the further description herein.

In one specific aspect of the invention, a Nanobody of the invention or a compound, construct or polypeptide of the invention comprising at least one Nanobody of the invention may have an increased half-life, compared to the corresponding amino acid sequence of the invention. Some preferred, but non-limiting examples of such Nanobodies, compounds and polypeptides will become clear to the skilled person based on the further disclosure herein, and for example comprise Nanobodies sequences or polypeptides of the invention that have been chemically modified to increase the half-life thereof (for example, by means of pegylation); amino acid sequences of the invention that comprise at least one additional binding site for binding to a serum protein (such as serum albumin, see for example EP 0 368 684 B1, page 4); or polypeptides of the invention that comprise at least one Nanobody of the invention that is linked to at least one moiety (and in particular at least one amino acid sequence) that increases the half-life of the Nanobody of the invention. Examples of polypeptides of the invention that comprise such half-life extending moieties or amino acid sequences will become clear to the skilled person based on the further disclosure herein; and for example include, without limitation, polypeptides in which the one or more Nanobodies of the invention are suitable linked to one or more serum proteins or fragments thereof (such as serum albumin or suitable fragments thereof) or to one or more binding units that can bind to serum proteins (such as, for example, Nanobodies or (single) domain antibodies that can bind to serum proteins such as serum albumin, serum immunoglobulins such as IgG, or transferrin); polypeptides in which a Nanobody of the invention is linked to an Fc portion (such as a human Fc) or a suitable part or fragment thereof; or polypeptides in which the one or more Nanobodies of the invention are suitable linked to one or more small proteins or peptides that can bind to serum proteins (such as, without limitation, the proteins and peptides described in WO 91/01743, WO 01/45746, WO 02/076489 and to the US provisional application of Ablynx N.V. entitled “Peptides capable of binding to serum proteins” of Ablynx N. V. filed on Dec. 5, 2006 (see also PCT/EP/2007/063348).

Again, as will be clear to the skilled person, such Nanobodies, compounds, constructs or polypeptides may contain one or more additional groups, residues, moieties or binding units, such as one or more further amino acid sequences and in particular one or more additional Nanobodies (i.e. not directed against a metalloproteinase from the ADAM family), so as to provide a tri- of multispecific Nanobody construct.

Generally, the Nanobodies of the invention (or compounds, constructs or polypeptides comprising the same) with increased half-life preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding amino acid sequence of the invention per se. For example, the Nanobodies, compounds, constructs or polypeptides of the invention with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding amino acid sequence of the invention per se.

In a preferred, but non-limiting aspect of the invention, such Nanobodies, compound, constructs or polypeptides of the invention exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, compounds or polypeptides of the invention may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

In another one aspect of the invention, a polypeptide of the invention comprises one or more (such as two or preferably one) Nanobodies of the invention linked (optionally via one or more suitable linker sequences) to one or more (such as two and preferably one) amino acid sequences that allow the resulting polypeptide of the invention to cross the blood brain barrier. In particular, said one or more amino acid sequences that allow the resulting polypeptides of the invention to cross the blood brain barrier may be one or more (such as two and preferably one) Nanobodies, such as the Nanobodies described in WO 02/057445, of which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO 06/040154) are preferred examples.

In particular, polypeptides comprising one or more Nanobodies of the invention are preferably such that they:

-   -   bind to a metalloproteinase from the ADAM family with a         dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or         less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more         preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association         constant (K_(A)) of 10⁵ to 10¹² liter/ moles or more, and         preferably 10⁷ to 10¹² liter/moles or more and more preferably         10⁸ to 10¹² liter/moles);         and/or such that they:     -   bind to a metalloproteinase from the ADAM family with a         k_(on)-rate of between 10²M⁻¹ s⁻¹ to about 10⁷M⁻¹ s⁻¹,         preferably between 10³M⁻¹ s⁻¹ and 10⁷M⁻¹ s⁻¹, more preferably         between 10⁴M⁻¹ s⁻¹ and 10⁷M⁻¹ s⁻¹, such as between 10⁵M⁻¹s⁻¹ and         10⁷M⁻¹ s⁻¹;         and/or such that they:     -   bind to a metalloproteinase from the ADAM family with a k_(off)         rate between 1 s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a         near irreversible complex with a t_(1/2) of multiple days),         preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably         between 10⁻³ s⁻¹ and 10⁻⁴ s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶         s⁻¹.

Preferably, a polypeptide that contains only one amino acid sequence of the invention is preferably such that it will bind to a metalloproteinase from the ADAM family with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. In this respect, it will be clear to the skilled person that a polypeptide that contains two or more Nanobodies of the invention may bind to a metalloproteinase from the ADAM family with an increased avidity, compared to a polypeptide that contains only one amino acid sequence of the invention.

Some preferred IC₅₀ values for binding of the amino acid sequences or polypeptides of the invention to a metalloproteinase from the ADAM family will become clear from the further description and examples herein.

Another aspect of this invention relates to a nucleic acid that encodes an amino acid sequence of the invention (such as a Nanobody of the invention) or a polypeptide of the invention comprising the same. Again, as generally described herein for the nucleic acids of the invention, such a nucleic acid may be in the form of a genetic construct, as defined herein.

In another aspect, the invention relates to host or host cell that expresses or that is capable of expressing an amino acid sequence (such as a Nanobody) of the invention and/or a polypeptide of the invention comprising the same; and/or that contains a nucleic acid of the invention. Some preferred but non-limiting examples of such hosts or host cells will become clear from the further description herein.

Another aspect of the invention relates to a product or composition containing or comprising at least one amino acid sequence of the invention, at least one polypeptide of the invention and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se, i.e. depending on the intended use of the composition. Such a product or composition may for example be a pharmaceutical composition (as described herein), a veterinary composition or a product or composition for diagnostic use (as also described herein). Some preferred but non-limiting examples of such products or compositions will become clear from the further description herein.

The invention further relates to methods for preparing or generating the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein. Some preferred but non-limiting examples of such methods will become clear from the further description herein.

The invention further relates to applications and uses of the amino acid sequences, compounds, constructs, polypeptides, nucleic acids, host cells, products and compositions described herein, as well as to methods for the prevention and/or treatment for diseases and disorders associated with a metalloproteinase from the ADAM family. Some preferred but non-limiting applications and uses will become clear from the further description herein.

Other aspects, embodiments, advantages and applications of the invention will also become clear from the further description hereinbelow.

Generally, it should be noted that the term Nanobody as used herein in its broadest sense is not limited to a specific biological source or to a specific method of preparation. For example, as will be discussed in more detail below, the Nanobodies of the invention can generally be obtained: (1) by isolating the V_(HH) domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a naturally occurring V_(HH) domain; (3) by “humanization” (as described herein) of a naturally occurring V_(HH) domain or by expression of a nucleic acid encoding a such humanized V_(HH) domain; (4) by “camelization” (as described herein) of a naturally occurring V_(H) domain from any animal species, and in particular a from species of mammal, such as from a human being, or by expression of a nucleic acid encoding such a camelized V_(H) domain; (5) by “camelisation” of a “domain antibody” or “Dab” as described by Ward et al (supra), or by expression of a nucleic acid encoding such a camelized V_(H) domain; (6) by using synthetic or semi-synthetic techniques for preparing proteins, polypeptides or other amino acid sequences known per se; (7) by preparing a nucleic acid encoding a Nanobody using techniques for nucleic acid synthesis known per se, followed by expression of the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing. Suitable methods and techniques for performing the foregoing will be clear to the skilled person based on the disclosure herein and for example include the methods and techniques described in more detail herein.

One preferred class of Nanobodies corresponds to the V_(HH) domains of naturally occurring heavy chain antibodies directed against a metalloproteinase from the ADAM family. As further described herein, such V_(HH) sequences can generally be generated or obtained by suitably immunizing a species of Camelid with a metalloproteinase from the ADAM family (i.e. so as to raise an immune response and/or heavy chain antibodies directed against a metalloproteinase from the ADAM family), by obtaining a suitable biological sample from said Camelid (such as a blood sample, serum sample or sample of B-cells), and by generating V_(HH) sequences directed against a metalloproteinase from the ADAM family, starting from said sample, using any suitable technique known per se. Such techniques will be clear to the skilled person and/or are further described herein.

Alternatively, such naturally occurring V_(HH) domains against a metalloproteinase from the ADAM family, can be obtained from naïve libraries of Camelid V_(HH) sequences, for example by screening such a library using a metalloproteinase from the ADAM family, or at least one part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known per se. Such libraries and techniques are for example described in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively, improved synthetic or semi-synthetic libraries derived from naïve V_(HH) libraries may be used, such as V_(HH) libraries obtained from naïve V_(HH) libraries by techniques such as random mutagenesis and/or CDR shuffling, as for example described in WO 00/43507.

Thus, in another aspect, the invention relates to a method for generating Nanobodies, that are directed against a metalloproteinase from the ADAM family. In one aspect, said method at least comprises the steps of:

-   a) providing a set, collection or library of Nanobody sequences; and -   b) screening said set, collection or library of Nanobody sequences     for Nanobody sequences that can bind to and/or have affinity for a     metalloproteinase from the ADAM family;     and -   c) isolating the amino acid sequence(s) that can bind to and/or have     affinity for a metalloproteinase from the ADAM family.

In such a method, the set, collection or library of Nanobody sequences may be a naïve set, collection or library of Nanobody sequences; a synthetic or semi-synthetic set, collection or library of Nanobody sequences; and/or a set, collection or library of Nanobody sequences that have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library of Nanobody sequences may be an immune set, collection or library of Nanobody sequences, and in particular an immune set, collection or library of V_(HH) sequences, that have been derived from a species of Camelid that has been suitably immunized with a metalloproteinase from the ADAM family or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of Nanobody or V_(HH) sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) Nanobody sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to WO 03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

In another aspect, the method for generating Nanobody sequences comprises at least the steps of:

-   a) providing a collection or sample of cells derived from a species     of Camelid that express immunoglobulin sequences; -   b) screening said collection or sample of cells for (i) cells that     express an immunoglobulin sequence that can bind to and/or have     affinity for a metalloproteinase from the ADAM family; and (ii)     cells that express heavy chain antibodies, in which substeps (i)     and (ii) can be performed essentially as a single screening step or     in any suitable order as two separate screening steps, so as to     provide at least one cell that expresses a heavy chain antibody that     can bind to and/or has affinity for a metalloproteinase from the     ADAM family;     and -   c) either (i) isolating from said cell the Vim sequence present in     said heavy chain antibody; or (ii) isolating from said cell a     nucleic acid sequence that encodes the V_(HH) sequence present in     said heavy chain antibody, followed by expressing said V_(HH)     domain.

In the method according to this aspect, the collection or sample of cells may for example be a collection or sample of B-cells. Also, in this method, the sample of cells may be derived from a Camelid that has been suitably immunized with a metalloproteinase from the ADAM family or a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

The above method may be performed in any suitable manner, as will be clear to the skilled person. Reference is for example made to EP 0 542 810, WO 05/19824, WO 04/051268 and WO 04/106377. The screening of step b) is preferably performed using a flow cytometry technique such as FACS. For this, reference is for example made to Lieby et al., Blood, Vol. 97, No. 12, 3820. Particular reference is made to the so-called “Nanoclone™” technique described in International application WO 06/079372 by Ablynx N. V.

In another aspect, the method for generating an amino acid sequence directed against a metalloproteinase from the ADAM family may comprise at least the steps of:

-   a) providing a set, collection or library of nucleic acid sequences     encoding heavy chain antibodies or Nanobody sequences; -   b) screening said set, collection or library of nucleic acid     sequences for nucleic acid sequences that encode a heavy chain     antibody or a Nanobody sequence that can bind to and/or has affinity     for a metalloproteinase from the ADAM family;     and -   c) isolating said nucleic acid sequence, followed by expressing the     V_(HH) sequence present in said heavy chain antibody or by     expressing said Nanobody sequence, respectively.

In such a method, the set, collection or library of nucleic acid sequences encoding heavy chain antibodies or Nanobody sequences may for example be a set, collection or library of nucleic acid sequences encoding a naïve set, collection or library of heavy chain antibodies or V_(HH) sequences; a set, collection or library of nucleic acid sequences encoding a synthetic or semi-synthetic set, collection or library of Nanobody sequences; and/or a set, collection or library of nucleic acid sequences encoding a set, collection or library of Nanobody sequences that have been subjected to affinity maturation.

In a preferred aspect of this method, the set, collection or library of amino acid sequences may be an immune set, collection or library of nucleic acid sequences encoding heavy chain antibodies or V_(HH) sequences derived from a Camelid that has been suitably immunized with a metalloproteinase from the ADAM family or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part, fragment, region, domain, loop or other epitope thereof. In one particular aspect, said antigenic determinant may be an extracellular part, region, domain, loop or other extracellular epitope(s).

In the above methods, the set, collection or library of nucleotide sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) nucleotide sequences encoding amino acid sequences will be clear to the person skilled in the art, for example on the basis of the further disclosure herein. Reference is also made to WO 03/054016 and to the review by Hoogenboom in Nature Biotechnology, 23, 9, 1105-1116 (2005).

As will be clear to the skilled person, the screening step of the methods described herein can also be performed as a selection step. Accordingly the term “screening” as used in the present description can comprise selection, screening or any suitable combination of selection and/or screening techniques. Also, when a set, collection or library of sequences is used, it may contain any suitable number of sequences, such as 1, 2, 3 or about 5, 10, 50, 100, 500, 1000, 5000, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ or more sequences.

Also, one or more or all of the sequences in the above set, collection or library of amino acid sequences may be obtained or defined by rational, or semi-empirical approaches such as computer modelling techniques or biostatics or datamining techniques.

Furthermore, such a set, collection or library can comprise one, two or more sequences that are variants from one another (e.g. with designed point mutations or with randomized positions), compromise multiple sequences derived from a diverse set of naturally diversified sequences (e.g. an immune library)), or any other source of diverse sequences (as described for example in Hoogenboom et al, Nat Biotechnol 23:1105, 2005 and Binz et al, Nat Biotechnol 2005, 23:1247). Such set, collection or library of sequences can be displayed on the surface of a phage particle, a ribosome, a bacterium, a yeast cell, a mammalian cell, and linked to the nucleotide sequence encoding the amino acid sequence within these carriers. This makes such set, collection or library amenable to selection procedures to isolate the desired amino acid sequences of the invention. More generally, when a sequence is displayed on a suitable host or host cell, it is also possible (and customary) to first isolate from said host or host cell a nucleotide sequence that encodes the desired sequence, and then to obtain the desired sequence by suitably expressing said nucleotide sequence in a suitable host organism. Again, this can be performed in any suitable manner known per se, as will be clear to the skilled person.

Yet another technique for obtaining V_(HH) sequences or Nanobody sequences directed against a metalloproteinase from the ADAM family involves suitably immunizing a transgenic mammal that is capable of expressing heavy chain antibodies (i.e. so as to raise an immune response and/or heavy chain antibodies directed against a metalloproteinase from the ADAM family), obtaining a suitable biological sample from said transgenic mammal that contains (nucleic acid sequences encoding) said V_(HH) sequences or Nanobody sequences (such as a blood sample, serum sample or sample of B-cells), and then generating V_(HH) sequences directed against a metalloproteinase from the ADAM family, starting from said sample, using any suitable technique known per se (such as any of the methods described herein or a hybridoma technique). For example, for this purpose, the heavy chain antibody-expressing mice and the further methods and techniques described in WO 02/085945, WO 04/049794 and WO 06/008548 and Janssens et al., Proc. Natl. Acad. Sci. USA. 2006 Oct. 10;103(41):15130-5 can be used. For example, such heavy chain antibody expressing mice can express heavy chain antibodies with any suitable (single) variable domain, such as (single) variable domains from natural sources (e.g. human (single) variable domains, Camelid (single) variable domains or shark (single) variable domains), as well as for example synthetic or semi-synthetic (single) variable domains.

The invention also relates to the V_(HH) sequences or Nanobody sequences that are obtained by the above methods, or alternatively by a method that comprises the one of the above methods and in addition at least the steps of determining the nucleotide sequence or amino acid sequence of said V_(HH) sequence or Nanobody sequence; and of expressing or synthesizing said V_(HH) sequence or Nanobody sequence in a manner known per se, such as by expression in a suitable host cell or host organism or by chemical synthesis.

As mentioned herein, a particularly preferred class of Nanobodies of the invention comprises Nanobodies with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V_(HH) domain, but that has been “humanized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring V_(HH) sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a V_(H) domain from a conventional 4-chain antibody from a human being (e.g. indicated above). This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein and the prior art on humanization referred to herein. Again, it should be noted that such humanized Nanobodies of the invention can be obtained in any suitable manner known per se (i.e. as indicated under points (1)-(8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring V_(HH) domain as a starting material.

Another particularly preferred class of Nanobodies of the invention comprises Nanobodies with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring V_(H) domain, but that has been “camelized”, i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring V_(H) domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a V_(HH) domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the further description herein. Such “camelizing” substitutions are preferably inserted at amino acid positions that form and/or are present at the V_(H)-V_(L) interface, and/or at the so-called Camelidae hallmark residues, as defined herein (see for example WO 94/04678 and Davies and Riechmann (1994 and 1996), supra). Preferably, the V_(H) sequence that is used as a starting material or starting point for generating or designing the camelized Nanobody is preferably a V_(H) sequence from a mammal, more preferably the V_(H) sequence of a human being, such as a V_(H)3 sequence. However, it should be noted that such camelized Nanobodies of the invention can be obtained in any suitable manner known per se (i.e. as indicated under points (1)-(8) above) and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring V_(H) domain as a starting material.

For example, again as further described herein, both “humanization” and “camelization” can be performed by providing a nucleotide sequence that encodes a naturally occurring V_(HH) domain or V_(H) domain, respectively, and then changing, in a manner known per se, one or more codons in said nucleotide sequence in such a way that the new nucleotide sequence encodes a “humanized” or “camelized” Nanobody of the invention, respectively. This nucleic acid can then be expressed in a manner known per se, so as to provide the desired Nanobody of the invention. Alternatively, based on the amino acid sequence of a naturally occurring V_(HH) domain or V_(H) domain, respectively, the amino acid sequence of the desired humanized or camelized Nanobody of the invention, respectively, can be designed and then synthesized de novo using techniques for peptide synthesis known per se. Also, based on the amino acid sequence or nucleotide sequence of a naturally occurring V_(HH) domain or V_(H) domain, respectively, a nucleotide sequence encoding the desired humanized or camelized Nanobody of the invention, respectively, can be designed and then synthesized de novo using techniques for nucleic acid synthesis known per se, after which the nucleic acid thus obtained can be expressed in a manner known per se, so as to provide the desired Nanobody of the invention.

Other suitable methods and techniques for obtaining the Nanobodies of the invention and/or nucleic acids encoding the same, starting from naturally occurring V_(H) sequences or preferably V_(HH) sequences, will be clear from the skilled person, and may for example comprise combining one or more parts of one or more naturally occurring V_(H) sequences (such as one or more FR sequences and/or CDR sequences), one or more parts of one or more naturally occurring V_(HH) sequences (such as one or more FR sequences or CDR sequences), and/or one or more synthetic or semi-synthetic sequences, in a suitable manner, so as to provide a Nanobody of the invention or a nucleotide sequence or nucleic acid encoding the same (which may then be suitably expressed). Nucleotide sequences encoding framework sequences of V_(HH) sequences or Nanobodies will be clear to the skilled person based on the disclosure herein and/or the further prior art cited herein (and/or may alternatively be obtained by PCR starting from the nucleotide sequences obtained using the methods described herein) and may be suitably combined with nucleotide sequences that encode the desired CDR's (for example, by PCR assembly using overlapping primers), so as to provide a nucleic acid encoding a Nanobody of the invention.

As mentioned herein, Nanobodies may in particular be characterized by the presence of one or more “Hallmark residues” (as described herein) in one or more of the framework sequences.

Thus, according to one preferred, but non-limiting aspect of the invention, a Nanobody in its broadest sense can be generally defined as a polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework     regions/sequences interrupted by three complementarity determining     regions/sequences, in which the amino acid residue at position 108     according to the Kabat numbering is Q;     and/or: -   b) an amino acid sequence that is comprised of four framework     regions/sequences interrupted by three complementarity determining     regions/sequences, in which the amino acid residue at position 45     according to the Kabat numbering is a charged amino acid (as defined     herein) or a cysteine residue, and position 44 is preferably an E;     and/or: -   c) an amino acid sequence that is comprised of four framework     regions/sequences interrupted by three complementarity determining     regions/sequences, in which the amino acid residue at position 103     according to the Kabat numbering is chosen from the group consisting     of P, R and S, and is in particular chosen from the group consisting     of R and S.

Thus, in a first preferred, but non-limiting aspect, a Nanobody of the invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which

-   a) the amino acid residue at position 108 according to the Kabat     numbering is Q; and/or in which:

-   b) the amino acid residue at position 45 according to the Kabat     numbering is a charged amino acid or a cysteine and the amino acid     residue at position 44 according to the Kabat numbering is     preferably E;     and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of P, R and S, and is     in particular chosen from the group consisting of R and S;     and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In particular, a Nanobody in its broadest sense can be generally defined as a polypeptide comprising:

-   a) an amino acid sequence that is comprised of four framework     regions/sequences interrupted by three complementarity determining     regions/sequences, in which the amino acid residue at position 108     according to the Kabat numbering is Q; and/or: -   b) an amino acid sequence that is comprised of four framework     regions/sequences interrupted by three complementarity determining     regions/sequences, in which the amino acid residue at position 44     according to the Kabat numbering is E and in which the amino acid     residue at position 45 according to the Kabat numbering is an R;     and/or: -   c) an amino acid sequence that is comprised of four framework     regions/sequences interrupted by three complementarity determining     regions/sequences, in which the amino acid residue at position 103     according to the Kabat numbering is chosen from the group consisting     of P, R and S, and is in particular chosen from the group consisting     of R and S.

Thus, according to a preferred, but non-limiting aspect, a Nanobody of the invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which

-   a) the amino acid residue at position 108 according to the Kabat     numbering is Q;     and/or in which:

-   b) the amino acid residue at position 44 according to the Kabat     numbering is E and in which the amino acid residue at position 45     according to the Kabat numbering is an R;     and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of P, R and S, and is     in particular chosen from the group consisting of R and S;     and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In particular, a Nanobody against a metalloproteinase from the ADAM family according to the invention may have the structure:

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which

-   a) the amino acid residue at position 108 according to the Kabat     numbering is Q; and/or in which:

-   b) the amino acid residue at position 44 according to the Kabat     numbering is E and in which the amino acid residue at position 45     according to the Kabat numbering is an R;     and/or in which:

-   c) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of P, R and S, and is     in particular chosen from the group consisting of R and S;     and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In particular, according to one preferred, but non-limiting aspect of the invention, a Nanobody can generally be defined as a polypeptide comprising an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which;

-   a-1) the amino acid residue at position 44 according to the Kabat     numbering is chosen from the group consisting of A, G, E, D, G, Q,     R, S, L; and is preferably chosen from the group consisting of G, E     or Q; and -   a-2) the amino acid residue at position 45 according to the Kabat     numbering is chosen from the group consisting of L, R or C; and is     preferably chosen from the group consisting of L or R; and -   a-3) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of W, R or S; and is     preferably W or R, and is most preferably W; -   a-4) the amino acid residue at position 108 according to the Kabat     numbering is Q;     or in which: -   b-1) the amino acid residue at position 44 according to the Kabat     numbering is chosen from the group consisting of E and Q; and -   b-2) the amino acid residue at position 45 according to the Kabat     numbering is R; and -   b-3) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of W, R and S; and is     preferably W; -   b-4) the amino acid residue at position 108 according to the Kabat     numbering is chosen from the group consisting of Q and L; and is     preferably Q;     or in which: -   c-1) the amino acid residue at position 44 according to the Kabat     numbering is chosen from the group consisting of A, G, E, D, Q, R, S     and L; and is preferably chosen from the group consisting of G, E     and Q; and -   c-2) the amino acid residue at position 45 according to the Kabat     numbering is chosen from the group consisting of L, R and C; and is     preferably chosen from the group consisting of L and R; and -   c-3) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of P, R and S; and is     in particular chosen from the group consisting of R and S; and -   c-4) the amino acid residue at position 108 according to the Kabat     numbering is chosen from the group consisting of Q and L; is     preferably Q;     and in which -   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of the invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which:

-   a-1) the amino acid residue at position 44 according to the Kabat     numbering is chosen from the group consisting of A, G, E, D, G, Q,     R, S, L; and is preferably chosen from the group consisting of G, E     or Q;     and in which:

-   a-2) the amino acid residue at position 45 according to the Kabat     numbering is chosen from the group consisting of L, R or C; and is     preferably chosen from the group consisting of L or R;     and in which:

-   a-3) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of W, R or S; and is     preferably W or R, and is most preferably W;     and in which

-   a-4) the amino acid residue at position 108 according to the Kabat     numbering is Q;     and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of the invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which:

-   b-1) the amino acid residue at position 44 according to the Kabat     numbering is chosen from the group consisting of E and Q;     and in which:

-   b-2) the amino acid residue at position 45 according to the Kabat     numbering is R;     and in which:

-   b-3) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of W, R and S; and is     preferably W;     and in which:

-   b-4) the amino acid residue at position 108 according to the Kabat     numbering is chosen from the group consisting of Q and L; and is     preferably Q;     and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of the invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which:

-   c-1) the amino acid residue at position 44 according to the Kabat     numbering is chosen from the group consisting of A, G, E, D, Q, R, S     and L; and is preferably chosen from the group consisting of G, E     and Q;     and in which:

-   c-2) the amino acid residue at position 45 according to the Kabat     numbering is chosen from the group consisting of L, R and C; and is     preferably chosen from the group consisting of L and R;     and in which:

-   c-3) the amino acid residue at position 103 according to the Kabat     numbering is chosen from the group consisting of P, R and S; and is     in particular chosen from the group consisting of R and S;     and in which:

-   c-4) the amino acid residue at position 108 according to the Kabat     numbering is chosen from the group consisting of Q and L; is     preferably Q;     and in which:

-   d) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

Two particularly preferred, but non-limiting groups of the Nanobodies of the invention are those according to a) above; according to (a-1) to (a-4) above; according to b) above; according to (b-1) to (b-4) above; according to (c) above; and/or according to (c-1) to (c-4) above, in which either:

-   i) the amino acid residues at positions 44-47 according to the Kabat     numbering form the sequence GLEW (SEQ ID NO: 1054) (or a GLEW-like     sequence as described herein) and the amino acid residue at position     108 is Q;     or in which: -   ii) the amino acid residues at positions 43-46 according to the     Kabat numbering form the sequence KERE (SEQ ID NO: 1055) or KQRE     (SEQ ID NO: 1056) (or a KERE-like sequence as described) and the     amino acid residue at position 108 is Q or L, and is preferably Q.

Thus, in another preferred, but non-limiting aspect, a Nanobody of the invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which:         i) the amino acid residues at positions 44-47 according to the         Kabat numbering form the sequence GLEW (SEQ ID NO: 1054) (or a         GLEW-like sequence as defined herein) and the amino acid residue         at position 108 is Q;         and in which:

-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of the invention may have the structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which FR1 to FR4 refer to framework regions 1 to 4,         respectively, and in which CDR1 to CDR3 refer to the         complementarity determining regions 1 to 3, respectively, and in         which:

-   i) the amino acid residues at positions 43-46 according to the Kabat     numbering form the sequence KERE (SEQ ID NO: 1055) or KQRE (SEQ ID     NO: 1056) (or a KERE-like sequence) and the amino acid residue at     position 108 is Q or L, and is preferably Q;     and in which:

-   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In the Nanobodies of the invention in which the amino acid residues at positions 43-46 according to the Kabat numbering form the sequence KERE (SEQ ID NO: 1055) or KQRE (SEQ ID NO: 1056), the amino acid residue at position 37 is most preferably F. In the Nanobodies of the invention in which the amino acid residues at positions 44-47 according to the Kabat numbering form the sequence GLEW (SEQ ID NO: 1054), the amino acid residue at position 37 is chosen from the group consisting of Y, H, I, L, V or F, and is most preferably V.

Thus, without being limited hereto in any way, on the basis of the amino acid residues present on the positions mentioned above, the Nanobodies of the invention can generally be classified on the basis of the following three groups:

-   i) The “GLEW-group”: Nanobodies with the amino acid sequence GLEW     (SEQ ID NO: 1054) at positions 44-47 according to the Kabat     numbering and Q at position 108 according to the Kabat numbering. As     further described herein, Nanobodies within this group usually have     a V at position 37, and can have a W, P, R or S at position 103, and     preferably have a W at position 103. The GLEW group also comprises     some GLEW-like sequences such as those mentioned in Table A-3 below.     More generally, and without limitation, Nanobodies belonging to the     GLEW-group can be defined as Nanobodies with a G at position 44     and/or with a W at position 47, in which position 46 is usually E     and in which preferably position 45 is not a charged amino acid     residue and not cysteine; -   ii) The “KERE-group”: Nanobodies with the amino acid sequence KERE     (SEQ ID NO: 1055) or KQRE (SEQ ID NO: 1056) (or another KERE-like     sequence) at positions 43-46 according to the Kabat numbering and Q     or L at position 108 according to the Kabat numbering. As further     described herein, Nanobodies within this group usually have a F at     position 37, an L or F at position 47; and can have a W, P, R or S     at position 103, and preferably have a W at position 103. More     generally, and without limitation, Nanobodies belonging to the     KERE-group can be defined as Nanobodies with a K, Q or R at position     44 (usually K) in which position 45 is a charged amino acid residue     or cysteine, and position 47 is as further defined herein; -   iii) The “103 P, R, S-group”: Nanobodies with a P, R or S at     position 103. These Nanobodies can have either the amino acid     sequence GLEW (SEQ ID NO: 1054) at positions 44-47 according to the     Kabat numbering or the amino acid sequence KERE (SEQ ID NO: 1055) or     KQRE (SEQ ID NO: 1056) at positions 43-46 according to the Kabat     numbering, the latter most preferably in combination with an F at     position 37 and an L or an F at position 47 (as defined for the     KERE-group); and can have Q or L at position 108 according to the     Kabat numbering, and preferably have Q.

Also, where appropriate, Nanobodies may belong to (i.e. have characteristics of) two or more of these classes. For example, one specifically preferred group of Nanobodies has GLEW (SEQ ID NO: 1054) or a GLEW-like sequence at positions 44-47; P, R or S (and in particular R) at position 103; and Q at position 108 (which may be humanized to L).

More generally, it should be noted that the definitions referred to above describe and apply to Nanobodies in the form of a native (i.e. non-humanized) V_(HH) sequence, and that humanized variants of these Nanobodies may contain other amino acid residues than those indicated above (i.e. one or more humanizing substitutions as defined herein). For example, and without limitation, in some humanized Nanobodies of the GLEW-group or the 103 P, R, S-group, Q at position 108 may be humanized to 108L. As already mentioned herein, other humanizing substitutions (and suitable combinations thereof) will become clear to the skilled person based on the disclosure herein. In addition, or alternatively, other potentially useful humanizing substitutions can be ascertained by comparing the sequence of the framework regions of a naturally occurring V_(HH) sequence with the corresponding framework sequence of one or more closely related human V_(H) sequences, after which one or more of the potentially useful humanizing substitutions (or combinations thereof) thus determined can be introduced into said V_(HH) sequence (in any manner known per se, as further described herein) and the resulting humanized V_(HH) sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. In this way, by means of a limited degree of trial and error, other suitable humanizing substitutions (or suitable combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework regions of) a Nanobody may be partially humanized or fully humanized.

Thus, in another preferred, but non-limiting aspect, a Nanobody of the invention may be a Nanobody belonging to the GLEW-group (as defined herein), and in which CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

In another preferred, but non-limiting aspect, a Nanobody of the invention may be a Nanobody belonging to the KERE-group (as defined herein), and CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

Thus, in another preferred, but non-limiting aspect, a Nanobody of the invention may be a Nanobody belonging to the 103 P, R, S-group (as defined herein), and in which CDR1, CDR2 and CDR3 are as defined herein, and are preferably as defined according to one of the preferred aspects herein, and are more preferably as defined according to one of the more preferred aspects herein.

Also, more generally and in addition to the 108Q, 43E/44R and 103 P,R,S residues mentioned above, the Nanobodies of the invention can contain, at one or more positions that in a conventional V_(H) domain would form (part of) the V_(H)/V_(L) interface, one or more amino acid residues that are more highly charged than the amino acid residues that naturally occur at the same position(s) in the corresponding naturally occurring V_(H) sequence, and in particular one or more charged amino acid residues (as mentioned in Table A-2). Such substitutions include, but are not limited to, the GLEW-like sequences mentioned in Table A-3 below; as well as the substitutions that are described in the International Application WO 00/29004 for so-called “microbodies”, e.g. so as to obtain a Nanobody with Q at position 108 in combination with KLEW (SEQ ID NO: 1087) at positions 44-47. Other possible substitutions at these positions will be clear to the skilled person based upon the disclosure herein.

In one aspect of the Nanobodies of the invention, the amino acid residue at position 83 is chosen from the group consisting of L, M, S, V and W; and is preferably L.

Also, in one aspect of the Nanobodies of the invention, the amino acid residue at position 83 is chosen from the group consisting of R, K, N, E, G, I, T and Q; and is most preferably either K or E (for Nanobodies corresponding to naturally occurring V_(HH) domains) or R (for “humanized” Nanobodies, as described herein). The amino acid residue at position 84 is chosen from the group consisting of P, A, R, S, D T, and V in one aspect, and is most preferably P (for Nanobodies corresponding to naturally occurring V_(HH) domains) or R (for “humanized” Nanobodies, as described herein).

Furthermore, in one aspect of the Nanobodies of the invention, the amino acid residue at position 104 is chosen from the group consisting of G and D; and is most preferably G.

Collectively, the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108, which in the Nanobodies are as mentioned above, will also be referred to herein as the “Hallmark Residues”. The Hallmark Residues and the amino acid residues at the corresponding positions of the most closely related human V_(H) domain, V_(H)3, are summarized in Table A-3.

Some especially preferred but non-limiting combinations of these Hallmark Residues as occur in naturally occurring V_(HH) domains are mentioned in Table A-4. For comparison, the corresponding amino acid residues of the human V_(H)3 called DP-47 have been indicated in italics.

TABLE A-3 Hallmark Residues in Nanobodies Position Human V_(H)3 Hallmark Residues  11 L, V; predominantly L L, M, S, V, W; preferably L  37 V, I, F; usually V F⁽¹⁾, Y, H, I, L or V, preferably F⁽¹⁾ or Y  44⁽⁸⁾ G G⁽²⁾, E⁽³⁾, A, D, Q, R, S, L; preferably G⁽²⁾, E⁽³⁾ or Q; most preferably G⁽²⁾ or E⁽³⁾.  45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, C, I, L, P, Q, V; preferably L⁽²⁾ or R⁽³⁾  47⁽⁸⁾ W, Y W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, M, R, S, V or Y; preferably W⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R  83 R or K; usually R R, K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, Q or T; preferably K or R; most preferably K  84 A, T, D; predominantly A P⁽⁵⁾, A, L, R, S, T, D, V; preferably P 103 W W⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S; preferably W 104 G G or D; preferably G 108 L, M or T; predominantly L Q, L⁽⁷⁾ or R; preferably Q or L⁽⁷⁾ Notes: ⁽¹⁾In particular, but not exclusively, in combination with KERE (SEQ ID NO: 1055) or KQRE (SEQ ID NO: 1056) at positions 43-46. ⁽²⁾Usually as GLEW (SEQ ID NO: 1054) at positions 44-47. ⁽³⁾Usually as KERE (SEQ ID NO: 1055) or KQRE (SEQ ID NO: 1056) at positions 43-46, e.g. as KEREL (SEQ ID NO: 1057), KEREF (SEQ ID NO: 1058), KQREL (SEQ ID NO: 1059), KQREF (SEQ ID NO: 1060) or KEREG (SEQ ID NO: 1061) at positions 43-47. Alternatively, also sequences such as TERE (SEQ ID NO: 1062) (for example TEREL (SEQ ID NO: 1063)), KECE (SEQ ID NO: 1064) (for example KECEL (SEQ ID NO: 1065) or KECER (SEQ ID NO:1066)), RERE (SEQ ID NO: 1067) (for example REREG (SEQ ID NO: 1068)), QERE (SEQ ID NO: 1069) (for example QEREG (SEQ ID NO: 1070)), KGRE (SEQ ID NO: 1071) (for example KGREG (SEQ ID NO: 1072)), KDRE (SEQ ID NO: 1073) (for example KDREV (SEQ ID NO: 1074)) are possible. Some other possible, but less preferred sequences include for example DECKL (SEQ ID NO: 1075) and NVCEL (SEQ ID NO: 1076). ⁽⁴⁾With both GLEW (SEQ ID NO: 1054) at positions 44-47 and KERE (SEQ ID NO: 1055) or KQRE (SEQ ID NO: 1056) at positions 43-46. ⁽⁵⁾Often as KP or EP at positions 83-84 of naturally occurring V_(HH) domains. ⁽⁶⁾In particular, but not exclusively, in combination with GLEW (SEQ ID NO: 1054) at positions 44-47. ⁽⁷⁾With the proviso that when positions 44-47 are GLEW (SEQ ID NO: 1054), position 108 is always Q in (non-humanized) V_(HH) sequences that also contain a W at 103. ⁽⁸⁾The GLEW group also contains GLEW-like sequences at positions 44-47, such as for example GVEW (SEQ ID NO: 1077), EPEW (SEQ ID NO: 1078), GLER (SEQ ID NO: 1079), DQEW (SEQ ID NO: 1080), DLEW (SEQ ID NO: 1081), GIEW (SEQ ID NO: 1082), ELEW (SEQ ID NO: 1083), GPEW (SEQ ID NO: 1084), EWLP (SEQ ID NO: 1085), GPER and (SEQ ID NO: 1086).

TABLE A-4 Some preferred but non-limiting combinations of Hallmark Residues in naturally occurring Nanobodies. 11 37 44 45 47 83 84 103 104 108 DP-47 (human) M V G L W R A W G L “KERE” group L F E R L K P W G Q L F E R F E P W G Q L F E R F K P W G Q L Y Q R L K P W G Q L F L R V K P Q G Q L F Q R L K P W G Q L F E R F K P W G Q “GLEW” group L V G L W K S W G Q M V G L W K P R G Q For humanization of these combinations, reference is made to the specification.

In the Nanobodies, each amino acid residue at any other position than the Hallmark Residues can be any amino acid residue that naturally occurs at the corresponding position (according to the Kabat numbering) of a naturally occurring V_(HH) domain.

Such amino acid residues will be clear to the skilled person. Tables A-5 to A-8 mention some non-limiting residues that can be present at each position (according to the Kabat numbering) of the FR1, FR2, FR3 and FR4 of naturally occurring V_(HH) domains. For each position, the amino acid residue that most frequently occurs at each position of a naturally occurring V_(HH) domain (and which is the most preferred amino acid residue for said position in a Nanobody) is indicated in bold; and other preferred amino acid residues for each position have been underlined (note: the number of amino acid residues that are found at positions 26-30 of naturally occurring V_(HH) domains supports the hypothesis underlying the numbering by Chothia (supra) that the residues at these positions already form part of CDR1.)

In Tables A-5-A-8, some of the non-limiting residues that can be present at each position of a human V_(H)3 domain have also been mentioned. Again, for each position, the amino acid residue that most frequently occurs at each position of a naturally occurring human V_(H)3 domain is indicated in bold; and other preferred amino acid residues have been underlined.

For reference only, Tables A-5-A-8 also contain data on the V_(HH) entropy (“V_(HH) Ent.”) and V_(HH) variability (“V_(HH) Var.”) at each amino acid position for a representative sample of 1118 V_(HH) sequences (data kindly provided by David Lutje Hulsing and Prof. Theo Verrips of Utrecht University). The values for the V_(HH) entropy and the V_(HH) variability provide a measure for the variability and degree of conservation of amino acid residues between the 1118 V_(HH) sequences analyzed: low values (i.e. <1, such as <0.5) indicate that an amino acid residue is highly conserved between the V_(HH) sequences (i.e. little variability). For example, the G at position 8 and the G at position 9 have values for the V_(HH) entropy of 0.1 and 0 respectively, indicating that these residues are highly conserved and have little variability (and in case of position 9 is G in all 1118 sequences analysed), whereas for residues that form part of the CDR's generally values of 1.5 or more are found (data not shown). Note that (1) the amino acid residues listed in the second column of Tables A-5-A-8 are based on a bigger sample than the 1118 V_(HH) sequences that were analysed for determining the V_(HH) entropy and V_(HH) variability referred to in the last two columns; and (2) the data represented below support the hypothesis that the amino acid residues at positions 27-30 and may be even also at positions 93 and 94 already form part of the CDR's (although the invention is not limited to any specific hypothesis or explanation, and as mentioned above, herein the numbering according to Kabat is used). For a general explanation of sequence entropy, sequence variability and the methodology for determining the same, see Oliveira et al., PROTEINS: Structure, Function and Genetics, 52: 544-552 (2003).

TABLE A-5 Non-limiting examples of amino acid residues in FR1 (for the footnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH) V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 1 E, Q Q, A, E — — 2 V V 0.2 1 3 Q Q, K 0.3 2 4 L L 0.1 1 5 V, L Q, E, L, V 0.8 3 6 E E, D, Q, A 0.8 4 7 S, T S, F 0.3 2 8 G, R G 0.1 1 9 G G 0 1 10 G, V G, D, R 0.3 2 11 Hallmark residue: L, M, S, V, W; preferably L 0.8 2 12 V, I V, A 0.2 2 13 Q, K, R Q, E, K, P, R 0.4 4 14 P A, Q, A, G, P, S, T, V 1 5 15 G G 0 1 16 G, R G, A, E, D 0.4 3 17 S S, F 0.5 2 18 L L, V 0.1 1 19 R, K R, K, L, N, S, T 0.6 4 20 L L, F, I, V 0.5 4 21 S S, A, F, T 0.2 3 22 C C 0 1 23 A, T A, D, E, P, S, T, V 1.3 5 24 A A, I, L, S, T, V 1 6 25 S S, A, F, P, T 0.5 5 26 G G, A, D, E, R, S, T, V 0.7 7 27 F S, F, R, L, P, G, N, 2.3 13 28 T N, T, E, D, S, I, R, A, G, R, F, Y 1.7 11 29 F, V F, L, D, S, I, G, V, A 1.9 11 30 S, D, G N, S, E, G, A, D, M, T 1.8 11

TABLE A-6 Non-limiting examples of amino acid residues in FR2 (for the footnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH) V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 36 W W 0.1 1 37 Hallmark residue: F⁽¹⁾, H, I, L, Y or V, preferably F⁽¹⁾ or Y 1.1 6 38 R R 0.2 1 39 Q Q, H, P, R 0.3 2 40 A A, F, G, L, P, T, V 0.9 7 41 P, S, T P, A, L, S 0.4 3 42 G G, E 0.2 2 43 K K, D, E, N, Q, R, T, V 0.7 6 44 Hallmark residue: G⁽²⁾, E⁽³⁾, A, D, Q, R, S, L; preferably G⁽²⁾, E⁽³⁾ or 1.3 5 Q; most preferably G⁽²⁾ or E⁽³⁾ 45 Hallmark residue: L⁽²⁾, R⁽³⁾, C, I, L, P, Q, V; preferably L⁽²⁾ or R⁽³⁾ 0.6 4 46 E, V E, D, K, Q, V 0.4 2 47 Hallmark residue: W⁽²⁾, L⁽¹⁾ or F⁽¹⁾, A, G, I, M, R, S, V or Y; 1.9 9 preferably W⁽²⁾, L⁽¹⁾, F⁽¹⁾ or R 48 V V, I, L 0.4 3 49 S, A, G A, S, G, T, V 0.8 3

TABLE A-7 Non-limiting examples of amino acid residues in FR3 (for the footnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH) V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 66 R R 0.1 1 67 F F, L, V 0.1 1 68 T T, A, N, S 0.5 4 69 I I, L, M, V 0.4 4 70 S S, A, F, T 0.3 4 71 R R, G, H, I, L, K, Q, S, T, W 1.2 8 72 D, E D, E, G, N, V 0.5 4 73 N, D, G N, A, D, F, I, K, L, R, S, T, V, Y 1.2 9 74 A, S A, D, G, N, P, S, T, V 1 7 75 K K, A, E, K, L, N, Q, R 0.9 6 76 N, S N, D, K, R, S, T, Y 0.9 6 77 S, T, I T, A, E, I, M, P, S 0.8 5 78 L, A V, L, A, F, G, I, M 1.2 5 79 Y, H Y, A, D, F, H, N, S, T 1 7 80 L L, F, V 0.1 1 81 Q Q, E, I, L, R, T 0.6 5 82 M M, I, L, V 0.2 2  82a N, G N, D, G, H, S, T 0.8 4  82b S S, N, D, G, R, T 1 6  82c L L, P, V 0.1 2 83 Hallmark residue: R, K⁽⁵⁾, N, E⁽⁵⁾, G, I, M, Q or T; preferably K or 0.9 7 R; most preferably K 84 Hallmark residue: P⁽⁵⁾, A, D, L, R, S, T, V; preferably P 0.7 6 85 E, G E, D, G, Q 0.5 3 86 D D 0 1 87 T, M T, A, S 0.2 3 88 A A, G, S 0.3 2 89 V, L V, A, D, I, L, M, N, R, T 1.4 6 90 Y Y, F 0 1 91 Y, H Y, D, F, H, L, S, T, V 0.6 4 92 C C 0 1 93 A, K, T A, N, G, H, K, N, R, S, T, V, Y 1.4 10 94 K, R, T A, V, C, F, G, I, K, L, R, S or T 1.6 9

TABLE A-8 Non-limiting examples of amino acid residues in FR4 (for the footnotes, see the footnotes to Table A-3) Amino acid residue(s): V_(HH) V_(HH) Pos. Human V_(H)3 Camelid V_(HH)'s Ent. Var. 103 Hallmark residue: W⁽⁴⁾, P⁽⁶⁾, R⁽⁶⁾, S; preferably W 0.4 2 104 Hallmark residue: G or D; preferably G 0.1 1 105 Q, R Q, E, K, P, R 0.6 4 106 G G 0.1 1 107 T T, A, I 0.3 2 108 Hallmark residue: Q, L⁽⁷⁾ or R; preferably Q or L⁽⁷⁾ 0.4 3 109 V V 0.1 1 110 T T, I, A 0.2 1 111 V V, A, I 0.3 2 112 S S, F 0.3 1 113 S S, A, L, P, T 0.4 3

Thus, in another preferred, but not limiting aspect, a Nanobody of the invention can be defined as an amino acid sequence with the (general) structure

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:

-   i) one or more of the amino acid residues at positions 11, 37, 44,     45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering     are chosen from the Hallmark residues mentioned in Table A-3;     and in which: -   ii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_(HH) sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein.

In particular, a Nanobody of the invention can be an amino acid sequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which:

-   i) (preferably) one or more of the amino acid residues at positions     11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat     numbering are chosen from the Hallmark residues mentioned in Table     A-3 (it being understood that V_(HH) sequences will contain one or     more Hallmark residues; and that partially humanized Nanobodies will     usually, and preferably, [still] contain one or more Hallmark     residues [although it is also within the scope of the invention to     provide—where suitable in accordance with the invention—partially     humanized Nanobodies in which all Hallmark residues, but not one or     more of the other amino acid residues, have been humanized]; and     that in fully humanized Nanobodies, where suitable in accordance     with the invention, all amino acid residues at the positions of the     Hallmark residues will be amino acid residues that occur in a human     V_(H)3 sequence. As will be clear to the skilled person based on the     disclosure herein that such V_(HH) sequences, such partially     humanized Nanobodies with at least one Hallmark residue, such     partially humanized Nanobodies without Hallmark residues and such     fully humanized Nanobodies all form aspects of this invention);     and in which: -   ii) said amino acid sequence has at least 80% amino acid identity     with at least one of the amino acid sequences of SEQ ID NO's: 1 to     22, in which for the purposes of determining the degree of amino     acid identity, the amino acid residues that form the CDR sequences     (indicated with X in the sequences of SEQ ID NO's: 1 to 22) are     disregarded;     and in which: -   iii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably     as defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_(HH) sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein.

TABLE A-9 Representative amino acid sequences for Nanobodies of the KERE, GLEW and P,R,S 103 group. KERE sequence no. 1 SEQ ID NO: 1 EVQLVESGGGLVQPGGSLRLSCAASGIPFSXXXXXWFRQAPGKQRDSVAXXXXXRFTI SRDNAKNTVYLQMNSLKPEDTAVYRCYFXXXXXWGQGTQVTVSS KERE sequence no. 2 SEQ ID NO: 2 QVKLEESGGGLVQAGGSLRLSCVGSGRTFSXXXXXWFRLAPGKEREFVAXXXXXRFTI SRDTASNRGYLHMNNLTPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 3 SEQ ID NO: 3 AVQLVDSGGGLVQAGDSLKLSCALTGGAFTXXXXXWFRQTPGREREFVAXXXXXRFTI SRDNAKNMVYLRMNSLIPEDAAVYSCAAXXXXXWGQGTLVTVSS KERE sequence no. 4 SEQ ID NO: 4 QVQLVESGGGLVEAGGSLRLSCTASESPFRXXXXXWFRQTSGQEREFVAXXXXXRFTI SRDDAKNTVWLHGSTLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 5 SEQ ID NO: 5 AVQLVESGGGLVQGGGSLRLACAASERIFDXXXXXWYRQGPGNERELVAXXXXXRFTI SMDYTKQTVYLHMNSLRPEDTGLYYCKIXXXXXWGQGTQVTVSS KERE sequence no. 6 SEQ ID NO: 6 DVKFVESGGGLVQAGGSLRLSCVASGFNFDXXXXXWFRQAPGKEREEVAXXXXXRFTI SSEKDKNSVYLQMNSLKPEDTALYICAGXXXXXWGRGTQVTVSS KERE sequence no. 7 SEQ ID NO: 7 QVRLAESGGGLVQSGGSLRLSCVASGSTYTXXXXXWYRQYPGKQRALVAXXXXXRFTI ARDSTKDTFCLQMNNLKPEDTAVYYCYAXXXXXWGQGTQVTVSS KERE sequence no. 8 SEQ ID NO: 8 EVQLVESGGGLVQAGGSLRLSCAASGFTSDXXXXXWFRQAPGKPREGVSXXXXXRFTI STDNAKNTVHLLMNRVNAEDTALYYCAVXXXXXWGRGTRVTVSS KERE sequence no. 9 SEQ ID NO: 9 QVQLVESGGGLVQPGGSLRLSCQASGDISTXXXXXWYRQVPGKLREFVAXXXXXRFTI SGDNAKRAIYLQMNNLKPDDTAVYYCNRXXXXXWGQGTQVTVSP KERE sequence no. 10 SEQ ID NO: 10 QVPVVESGGGLVQAGDSLRLFCAVPSFTSTXXXXXWFRQAPGKEREFVAXXXXXRFTI SRNATKNTLTLRMDSLKPEDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 11 SEQ ID NO: 11 EVQLVESGGGLVQAGDSLRLFCTVSGGTASXXXXXWFRQAPGEKREFVAXXXXXRFTI ARENAGNMVYLQMNNLKPDDTALYTCAAXXXXXWGRGTQVTVSS KERE sequence no. 12 SEQ ID NO:12 AVQLVESGGDSVQPGDSQTLSCAASGRTNSXXXXXWFRQAPGKERVFLAXXXXXRFTI SRDSAKNMMYLQMNNLKPQDTAVYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 13 SEQ ID NO:13 AVQLVESGGGLVQAGGSLRLSCVVSGLTSSXXXXXWFRQTPWQERDFVAXXXXXRFTI ISRDNYKDTVLLEMNFLKPEDTAIYYCAAXXXXXWGQGTQVTVSS KERE sequence no. 14 SEQ ID NO: 14 AVQLVESGGGLVQAGASLRLSCATSTRTLDXXXXXWFRQAPGRDREFVAXXXXXRFTI VSRDSAENTVALQMNSLKPEDTAVYYCAAXXXXXWGQGTRVTVSS KERE sequence no. 15 SEQ ID NO: 15 QVQLVESGGGLVQPGGSLRLSCTVSRLTAHXXXXXWFRQAPGKEREAVSXXXXXRFTI SRDYAGNTAFLQMDSLKPEDTGVYYCATXXXXXWGQGTQVTVSS KERE sequence no. 16 SEQ ID NO: 16 EVQLVESGGELVQAGGSLKLSCTASGRNFVXXXXXWFRRAPGKEREFVAXXXXXRFT VSRDNGKNTAYLRMNSLKPEDTADYYCAVXXXXXLGSGTQVTVSS GLEW sequence no. 1 SEQ ID NO: 17 AVQLVESGGGLVQPGGSLRLSCAASGFTFSXXXXXWVRQAPGKVLEWVSXXXXXRFTI SRDNAKNTLYLQMNSLKPEDTAVYYCVKXXXXXGSQGTQVTVSS GLEW sequence no. 2 SEQ ID NO: 18 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRF KISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS GLEW sequence no. 3 SEQ ID NO: 19 EVQLVESGGGLALPGGSLTLSCVFSGSTFSXXXXXWVRHTPGKAEEWVSXXXXXRFTI SRDNAKNTLYLEMNSLSPEDTAMYYCGRXXXXXRSKGIQVTVSS P, R, S 103 sequence no. 1 SEQ ID NO: 20 AVQLVESGGGLVQAGGSLRLSCAASGRTFSXXXXXWFRQAPGKEREFVAXXXXXRFTI SRDNAKNTVYLQMNSLKPEDTAVYYCAAXXXXXRGQGTQVTVSS P, R, S 103 sequence no. 2 SEQ ID NO: 21 DVQLVESGGDLVQPGGSLRLSCAASGFSFDXXXXXWLRQTPGKGLEWVGXXXXXRFT ISRDNAKNMLYLHLNNLKSEDTAVYYCRRXXXXXLGQGTQVTVSS P, R, S 103 sequence no. 3 SEQ ID NO: 22 EVQLVESGGGLVQPGGSLRLSCVCVSSGCTXXXXXWVRQAPGKAEEWVSXXXXXRF KISRDNAKKTLYLQMNSLGPEDTAMYYCQRXXXXXRGQGTQVTVSS The CDR's are indicated with XXXX

In particular, a Nanobody of the invention of the KERE group can be an amino acid sequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4         in which:

-   i) the amino acid residue at position 45 according to the Kabat     numbering is a charged amino acid (as defined herein) or a cysteine     residue, and position 44 is preferably an E;     and in which:

-   ii) FR1 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-10 Representative FW1 sequences for Nanobodies of the KERE-group. KERE FW1 sequence no. 1 SEQ ID NO: 23 QVQRVESGGGLVQAGGSLRLSCAASGRTSS KERE FW1 sequence no. 2 SEQ ID NO: 24 QVQLVESGGGLVQTGDSLSLSCSASGRTFS KERE FW1 sequence no. 3 SEQ ID NO: 25 QVKLEESGGGLVQAGDSLRLSCAATGRAFG KERE FW1 sequence no. 4 SEQ ID NO: 26 AVQLVESGGGLVQPGESLGLSCVASGRDFV KERE FW1 sequence no. 5 SEQ ID NO: 27 EVQLVESGGGLVQAGGSLRLSCEVLGRTAG KERE FW1 sequence no. 6 SEQ ID NO: 28 QVQLVESGGGWVQPGGSLRLSCAASETILS KERE FW1 sequence no. 7 SEQ ID NO: 29 QVQLVESGGGTVQPGGSLNLSCVASGNTFN KERE FW1 sequence no. 8 SEQ ID NO: 30 EVQLVESGGGLAQPGGSLQLSCSAPGFTLD KERE FW1 sequence no. 9 SEQ ID NO: 31 AQELEESGGGLVQAGGSLRLSCAASGRTFN and in which:

-   iii) FR2 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-11 Representative FW2 sequences for Nanobodies of the KERE-group. KERE FW2 SEQ ID NO: 41 WFRQAPGKEREFVA sequence no. 1 KERE FW2 SEQ ID NO: 42 WFRQTPGREREFVA sequence no. 2 KERE FW2 SEQ ID NO: 43 WYRQAPGKQREMVA sequence no. 3 KERE FW2 SEQ ID NO: 44 WYRQGPGKQRELVA sequence no. 4 KERE FW2 SEQ ID NO: 45 WIRQAPGKEREGVS sequence no. 5 KERE FW2 SEQ ID NO: 46 WFREAPGKEREGIS sequence no. 6 KERE FW2 SEQ ID NO: 47 WYRQAPGKERDLVA sequence no. 7 KERE FW2 SEQ ID NO: 48 WFRQAPGKQREEVS sequence no. 8 KERE FW2 SEQ ID NO: 49 WFRQPPGKVREFVG sequence no. 9 and in which:

-   iv) FR3 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-12 Representative FW3 sequences for Nanobodies of the KERE-group. KERE FW3 SEQ ID RFTISRDNAKNTVYLQMNSLKPEDTAVYRCYF sequence NO: 50 no. 1 KERE FW3 SEQ ID RFAISRDNNKNTGYLQMNSLEPEDTAVYYCAA sequence NO: 51 no. 2 KERE FW3 SEQ ID RFTVARNNAKNTVNLEMNSLKPEDTAVYYCAA sequence NO: 52 no. 3 KERE FW3 SEQ ID RFTISRDIAKNTVDLLMNNLEPEDTAVYYCAA sequence NO: 53 no. 4 KERE FW3 SEQ ID RLTISRDNAVDTMYLQMNSLKPEDTAVYYCAA sequence NO: 54 no. 5 KERE FW3 SEQ ID RFTISRDNAKNTVYLQMDNVKPEDTAIYYCAA sequence NO: 55 no. 6 KERE FW3 SEQ ID RFTISKDSGKNTVYLQMTSLKPEDTAVYYCAT sequence NO: 56 no. 7 KERE FW3 SEQ ID RFTISRDSAKNMMYLQMNNLKPQDTAVYYCAA sequence NO: 57 no. 8 KERE FW3 SEQ ID RFTISRENDKSTVYLQLNSLKPEDTAVYYCAA sequence NO: 58 no. 9 KERE FW3 SEQ ID RFTISRDYAGNTAYLQMNSLKPEDTGVYYCAT sequence NO: 59 no. 10 and in which:

-   v) FR4 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-13 Representative FW4 sequences for Nanobodies of the KERE-group. KERE FW4 sequence no. 1 SEQ ID NO: 60 WGQGTQVTVSS KERE FW4 sequence no. 2 SEQ ID NO: 61 WGKGTLVTVSS KERE FW4 sequence no. 3 SEQ ID NO: 62 RGQGTRVTVSS KERE FW4 sequence no. 4 SEQ ID NO: 63 WGLGTQVTISS and in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_(HH) sequences or partially humanized Nanobodies).

Also, the above Nanobodies may for example be V_(HH) sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_(HH) sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein. With regard to framework 1, it will be clear to the skilled person that, when an amino acid sequence as outlined above is generated by expression of a nucleotide sequence, the first four amino acid sequences (i.e. amino acid residues 1-4 according to the Kabat numbering) may often be determined by the primer(s) that have been used to generate said nucleic acid. Thus, for determining the degree of amino acid identity, the first four amino acid residues are preferably disregarded.

Also, with regard to framework 1, and although amino acid positions 27 to 30 are according to the Kabat numbering considered to be part of the framework regions (and not the CDR's), it has been found by analysis of a database of more than 1000 V_(HH) sequences that the positions 27 to 30 have a variability (expressed in terms of V_(HH) entropy and V_(HH) variability—see Tables A-5 to A-8) that is much greater than the variability on positions 1 to 26. Because of this, for determining the degree of amino acid identity, the amino acid residues at positions 27 to 30 are preferably also disregarded.

In view of this, a Nanobody of the KERE class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which:

-   i) the amino acid residue at position 45 according to the Kabat     numbering is a charged amino acid (as defined herein) or a cysteine     residue, and position 44 is preferably an E;     and in which: -   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the     Kabat numbering, has at least 80% amino acid identity with at least     one of the following amino acid sequences:

TABLE A-14 Representative FW1 sequences (amino acid residues 5 to 26) for Nanobodies of the KERE-group. KERE FW1 se- SEQ ID NO: 32 VESGGGLVQPGGSLRLSCAASG quence no. 10 KERE FW1 se- SEQ ID NO: 33 VDSGGGLVQAGDSLKLSCALTG quence no. 11 KERE FW1 se- SEQ ID NO: 34 VDSGGGLVQAGDSLRLSCAASG quence no. 12 KERE FW1 se- SEQ ID NO: 35 VDSGGGLVEAGGSLRLSCQVSE quence no. 13 KERE FW1 se- SEQ ID NO: 36 QDSGGGSVQAGGSLKLSCAASG quence no. 14 KERE FW1 se- SEQ ID NO: 37 VQSGGRLVQAGDSLRLSCAASE quence no. 15 KERE FW1 se- SEQ ID NO: 38 VESGGTLVQSGDSLKLSCASST quence no. 16 KERE FW1 se- SEQ ID NO: 39 MESGGDSVQSGGSLTLSCVASG quence no. 17 KERE FW1 se- SEQ ID NO: 40 QASGGGLVQAGGSLRLSCSASV quence no. 18 and in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4     of Nanobodies of the KERE-class;     and in which: -   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_(HH) sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein.

A Nanobody of the GLEW class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which

-   i) preferably, when the Nanobody of the GLEW-class is a     non-humanized Nanobody, the amino acid residue in position 108 is Q; -   ii) FR1 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-15 Representative FW1 sequences for Nanobodies of the GLEW-group. GLEW FW1 se- SEQ ID QVQLVESGGGLVQPGGSLRLSCAASGFTFS quence no. 1 NO: 64 GLEW FW1 se- SEQ ID EVHLVESGGGLVRPGGSLRLSCAAFGFIFK quence no. 2 NO: 65 GLEW FW1 se- SEQ ID QVKLEESGGGLAQPGGSLRLSCVASGFTFS quence no. 3 NO: 66 GLEW FW1 se- SEQ ID EVQLVESGGGLVQPGGSLRLSCVCVSSGCT quence no. 4 NO: 67 GLEW FW1 se- SEQ ID EVQLVESGGGLALPGGSLTLSCVFSGSTFS quence no. 5 NO: 68 and in which:

-   iii) FR2 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-16 Representative FW2 sequences for Nanobodies of the GLEW-group. GLEW FW2 sequence no. SEQ ID NO: 72 WVRQAPGKVLEWVS 1 GLEW FW2 sequence no. SEQ ID NO: 73 WVRRPPGKGLEWVS 2 GLEW FW2 sequence no. SEQ ID NO: 74 WVRQAPGMGLEWVS 3 GLEW FW2 sequence no. SEQ ID NO: 75 WVRQAPGKEPEWVS 4 GLEW FW2 sequence no. SEQ ID NO: 76 WVRQAPGKDQEWVS 5 GLEW FW2 sequence no. SEQ ID NO: 77 WVRQAPGKAEEWVS 6 GLEW FW2 sequence no. SEQ ID NO: 78 WVRQAPGKGLEWVA 7 GLEW FW2 sequence no. SEQ ID NO: 79 WVRQAPGRATEWVS 8 and in which:

-   iv) FR3 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-17 Representative FW3 sequences for Nanobodies of the GLEW-group. GLEW FW3 SEQ ID RFTISRDNAKNTLYLQMNSLKPEDTAVYYCVK sequence NO: 80 no. 1 GLEW FW3 SEQ ID RFTISRDNARNTLYLQMDSLIPEDTALYYCAR sequence NO: 81 no. 2 GLEW FW3 SEQ ID RFTSSRDNAKSTLYLQMNDLKPEDTALYYCAR sequence NO: 82 no. 3 GLEW FW3 SEQ ID RFIISRDNAKNTLYLQMNSLGPEDTAMYYCQR sequence NO: 83 no. 4 GLEW FW3 SEQ ID RFTASRDNAKNTLYLQMNSLKSEDTARYYCAR sequence NO: 84 no. 5 GLEW FW3 SEQ ID RFTISRDNAKNTLYLQMDDLQSEDTAMYYCGR sequence NO: 85 no. 6 and in which:

-   v) FR4 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-18 Representative FW4 sequences for Nanobodies of the GLEW-group. GLEW FW4 sequence no. 1 SEQ ID NO: 86 GSQGTQVTVSS GLEW FW4 sequence no. 2 SEQ ID NO: 87 LRGGTQVTVSS GLEW FW4 sequence no. 3 SEQ ID NO: 88 RGQGTLVTVSS GLEW FW4 sequence no. 4 SEQ ID NO: 89 RSRGIQVTVSS GLEW FW4 sequence no. 5 SEQ ID NO: 90 WGKGTQVTVSS GLEW FW4 sequence no. 6 SEQ ID NO: 91 WGQGTQVTVSS and in which:

-   vi) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_(HH) sequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled person that, for determining the degree of amino acid identity, the amino acid residues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the GLEW class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which:

-   i) preferably, when the Nanobody of the GLEW-class is a     non-humanized Nanobody, the amino acid residue in position 108 is Q;     and in which: -   ii) FR1 is an amino acid sequence that, on positions 5 to 26 of the     Kabat numbering, has at least 80% amino acid identity with at least     one of the following amino acid sequences:

TABLE A-19 Representative FW1 sequences (amino acid residues 5 to 26) for Nanobodies of the KERE-group. GLEW FW1 SEQ ID NO: 69 VESGGGLVQPGGSLRLSCAASG sequence no. 6 GLEW FW1 SEQ ID NO: 70 EESGGGLAQPGGSLRLSCVASG sequence no. 7 GLEW FW1 SEQ ID NO: 71 VESGGGLALPGGSLTLSCVFSG sequence no. 8 and in which:

-   iii) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4     of Nanobodies of the GLEW-class;     and in which: -   iv) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_(HH) sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein. In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_(HH) sequences or partially humanized Nanobodies).

A Nanobody of the P, R, S 103 class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which

-   i) the amino acid residue at position 103 according to the Kabat     numbering is different from W;     and in which: -   ii) preferably the amino acid residue at position 103 according to     the Kabat numbering is P, R or S, and more preferably R;     and in which: -   iii) FR1 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-20 Representative FW1 sequences for Nanobodies of the P, R, S 103-group. P, R, S 103 SEQ ID AVQLVESGGGLVQAGGSLRLSCAASGRTFS FW1 sequence NO: 92 no. 1 P, R, S 103 SEQ ID QVQLQESGGGMVQPGGSLRLSCAASGFDFG FW1 sequence NO: 93 no. 2 P, R, S 103 SEQ ID EVHLVESGGGLVRPGGSLRLSCAAFGFIFK FW1 sequence NO: 94 no. 3 P, R, S 103 SEQ ID QVQLAESGGGLVQPGGSLKLSCAASRTIVS FW1 sequence NO: 95 no. 4 P, R, S 103 SEQ ID QEHLVESGGGLVDIGGSLRLSCAASERIFS FW1 sequence NO: 96 no. 5 P, R, S 103 SEQ ID QVKLEESGGGLAQPGGSLRLSCVASGFTFS FW1 sequence NO: 97 no. 6 P, R, S 103 SEQ ID EVQLVESGGGLVQPGGSLRLSCVCVSSGCT FW1 sequence NO: 98 no. 7 P, R, S 103 SEQ ID EVQLVESGGGLALPGGSLTLSCVFSGSTFS FW1 sequence NO: 99 no. 8 and in which

-   iv) FR2 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-21 Representative FW2 sequences for Nanobodies of the P, R, S 103-group. P, R, S 103 FW2 SEQ ID NO: 102 WFRQAPGKEREFVA sequence no. 1 P, R, S 103 FW2 SEQ ID NO: 103 WVRQAPGKVLEWVS sequence no. 2 P, R, S 103 FW2 SEQ ID NO: 104 WVRRPPGKGLEWVS sequence no. 3 P, R, S 103 FW2 SEQ ID NO: 105 WIRQAPGKEREGVS sequence no. 4 P, R, S 103 FW2 SEQ ID NO: 106 WVRQYPGKEPEWVS sequence no. 5 P, R, S 103 FW2 SEQ ID NO: 107 WFRQPPGKEHEFVA sequence no. 6 P, R, S 103 FW2 SEQ ID NO: 108 WYRQAPGKRTELVA sequence no. 7 P, R, S 103 FW2 SEQ ID NO: 109 WLRQAPGQGLEWVS sequence no. 8 P, R, S 103 FW2 SEQ ID NO: 110 WLRQTPGKGLEWVG sequence no. 9 P, R, S 103 FW2 SEQ ID NO: 111 WVRQAPGKAEEFVS sequence no. 10 and in which:

-   v) FR3 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-22 Representative FW3 sequences for Nanobodies of the P, R, S 103-group. P, R, S SEQ ID RFTISRDNAKNTVYLQMNSLKPEDTAVYYCAA 103 FW3 NO: 112 sequence no. 1 P, R, S SEQ ID RFTISRDNARNTLYLQMDSLIPEDTALYYCAR 103 FW3 NO: 113 sequence no. 2 P, R, S SEQ ID RFTISRDNAKNEMYLQMNNLKTEDTGVYWCGA 103 FW3 NO: 114 sequence no. 3 P, R, S SEQ ID RFTISSDSNRNMIYLQMNNLKPEDTAVYYCAA 103 FW3 NO: 115 sequence no. 4 P, R, S SEQ ID RFTISRDNAKNMLYLHLNNLKSEDTAVYYCRR 103 FW3 NO: 116 sequence no. 5 P, R, S SEQ ID RFTISRDNAKKTVYLRLNSLNPEDTAVYSCNL 103 FW3 NO: 117 sequence no. 6 P, R, S SEQ ID RFKISRDNAKKTLYLQMNSLGPEDTAMYYCQR 103 FW3 NO: 118 sequence no. 7 P, R, S SEQ ID RFTVSRDNGKNTAYLRMNSLKPEDTADYYCAV 103 FW3 NO: 119 sequence no. 8 and in which:

-   vi) FR4 is an amino acid sequence that has at least 80% amino acid     identity with at least one of the following amino acid sequences:

TABLE A-23 Representative FW4 sequences for Nanobodies of the P, R, S 103-group. P, R, S 103 FW4 sequence SEQ ID NO: 120 RGQGTQVTVSS no. 1 P, R, S 103 FW4 sequence SEQ ID NO: 121 LRGGTQVTVSS no. 2 P, R, S 103 FW4 sequence SEQ ID NO: 122 GNKGTLVTVSS no. 3 P, R, S 103 FW4 sequence SEQ ID NO: 123 SSPGTQVTVSS no. 4 P, R, S 103 FW4 sequence SEQ ID NO: 124 SSQGTLVTVSS no. 5 P, R, S 103 FW4 sequence SEQ ID NO: 125 RSRGIQVTVSS no. 6 and in which:

-   vii) CDR1, CDR2 and CDR3 are as defined herein, and are preferably     as defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_(HH) sequences or partially humanized Nanobodies).

With regard to framework 1, it will again be clear to the skilled person that, for determining the degree of amino acid identity, the amino acid residues on positions 1 to 4 and 27 to 30 are preferably disregarded.

In view of this, a Nanobody of the P,R,S 103 class may be an amino acid sequence that is comprised of four framework regions/sequences interrupted by three complementarity determining regions/sequences, in which:

-   i) the amino acid residue at position 103 according to the Kabat     numbering is different from W;     and in which: -   ii) preferably the amino acid residue at position 103 according to     the Kabat numbering is P, R or S, and more preferably R;     and in which: -   iii) FR1 is an amino acid sequence that, on positions 5 to 26 of the     Kabat numbering, has at least 80% amino acid identity with at least     one of the following amino acid sequences:

TABLE A-24 Representative FW1 sequences (amino acid residues 5 to 26) for Nanobodies of the P, R, S 103-group. P, R,S 103 SEQ ID NO: 100 VESGGGLVQAGGSLRLSCAASG FW1 sequence no. 9 P, R,S 103 SEQ ID NO: 101 AESGGGLVQPGGSLKLSCAASR FW1 sequence no. 10 and in which:

-   iv) FR2, FR3 and FR4 are as mentioned herein for FR2, FR3 and FR4 of     Nanobodies of the P,R,S 103 class;     and in which: -   v) CDR1, CDR2 and CDR3 are as defined herein, and are preferably as     defined according to one of the preferred aspects herein, and are     more preferably as defined according to one of the more preferred     aspects herein.

The above Nanobodies may for example be V_(HH) sequences or may be humanized Nanobodies. When the above Nanobody sequences are V_(HH) sequences, they may be suitably humanized, as further described herein. When the Nanobodies are partially humanized Nanobodies, they may optionally be further suitably humanized, again as described herein.

In the above Nanobodies, one or more of the further Hallmark residues are preferably as described herein (for example, when they are V_(HH) sequences or partially humanized Nanobodies).

In another preferred, but non-limiting aspect, the invention relates to a Nanobody as described above, in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053. This degree of amino acid identity can for example be determined by determining the degree of amino acid identity (in a manner described herein) between said Nanobody and one or more of the sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053, in which the amino acid residues that form the framework regions are disregarded. Such Nanobodies can be as further described herein.

As already mentioned herein, another preferred but non-limiting aspect of the invention relates to a Nanobody with an amino acid sequence that is chosen from the group consisting of SEQ ID NO's: 868 to 973 and/or 1044 to 1053 or from the group consisting of from amino acid sequences that have more than 80%, preferably more than 90%, more preferably more than 95%, such as 99% or more sequence identity (as defined herein) with at least one of the amino acid sequences of SEQ ID NO's: 868 to 973 and/or 1044 to 1053.

Also, in the above Nanobodies:

-   i) any amino acid substitution (when it is not a humanizing     substitution as defined herein) is preferably, and compared to the     corresponding amino acid sequence of SEQ ID NO's: 868 to 973 and/or     1044 to 1053, a conservative amino acid substitution, (as defined     herein);     and/or: -   ii) its amino acid sequence preferably contains either only amino     acid substitutions, or otherwise preferably no more than 5,     preferably no more than 3, and more preferably only 1 or 2 amino     acid deletions or insertions, compared to the corresponding amino     acid sequence of SEQ ID NO's: 868 to 973 and/or 1044 to 1053;     and/or -   iii) the CDR's may be CDR's that are derived by means of affinity     maturation, for example starting from the CDR's of to the     corresponding amino acid sequence of SEQ ID NO's: 868 to 973 and/or     1044 to 1053.

Preferably, the CDR sequences and FR sequences in the Nanobodies of the invention are such that the Nanobodies of the invention (and polypeptides of the invention comprising the same):

-   -   bind to a metalloproteinase from the ADAM family with a         dissociation constant (K_(D)) of 10⁻⁵ to 10⁻¹² moles/liter or         less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or less and more         preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e. with an association         constant (K_(A)) of 10⁵ to 10¹² liter/moles or more, and         preferably 10⁷ to 10¹² liter/moles or more and more preferably         10⁸ to 10¹² liter/moles);         and/or such that they:     -   bind to a metalloproteinase from the ADAM family with a         k_(on)-rate of between 10²M⁻¹ s⁻¹ to about 10⁷M⁻¹ s⁻¹,         preferably between 10³M⁻¹ s⁻¹ and 10⁷M⁻¹ s⁻¹, more preferably         between 10⁴M⁻¹ s⁻¹ and 10⁷M⁻¹ s⁻¹, such as between 10⁵M⁻¹ s⁻¹         and 10⁷M⁻¹ s⁻¹;         and/or such that they:     -   bind to a metalloproteinase from the ADAM family with a k_(off)         rate between 1 s⁻¹ (t_(1/2)=0.69 s) and 10⁻⁶ s⁻¹ (providing a         near irreversible complex with a t_(1/2) of multiple days),         preferably between 10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably         between 10⁻³ s⁻¹ and 10⁻⁴s⁻¹, such as between 10⁻⁴ s⁻¹ and 10⁻⁶         s⁻¹.

Preferably, CDR sequences and FR sequences present in the Nanobodies of the invention are such that the Nanobodies of the invention will bind to a metalloproteinase from the ADAM family with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM.

According to one non-limiting aspect of the invention, a Nanobody may be as defined herein, but with the proviso that it has at least “one amino acid difference” (as defined herein) in at least one of the framework regions compared to the corresponding framework region of a naturally occurring human V_(H) domain, and in particular compared to the corresponding framework region of DP-47. More specifically, according to one non-limiting aspect of the invention, a Nanobody may be as defined herein, but with the proviso that it has at least “one amino acid difference” (as defined herein) at at least one of the Hallmark residues (including those at positions 108, 103 and/or 45) compared to the corresponding framework region of a naturally occurring human V_(H) domain, and in particular compared to the corresponding framework region of DP-47. Usually, a Nanobody will have at least one such amino acid difference with a naturally occurring V_(H) domain in at least one of FR2 and/or FR4, and in particular at at least one of the Hallmark residues in FR2 and/or FR4 (again, including those at positions 108, 103 and/or 45).

Also, a humanized Nanobody of the invention may be as defined herein, but with the proviso that it has at least “one amino acid difference” (as defined herein) in at least one of the framework regions compared to the corresponding framework region of a naturally occurring V_(HH) domain. More specifically, according to one non-limiting aspect of the invention, a humanized Nanobody may be as defined herein, but with the proviso that it has at least “one amino acid difference” (as defined herein) at at least one of the Hallmark residues (including those at positions 108, 103 and/or 45) compared to the corresponding framework region of a naturally occurring V_(HH) domain. Usually, a humanized Nanobody will have at least one such amino acid difference with a naturally occurring V_(HH) domain in at least one of FR2 and/or FR4, and in particular at at least one of the Hallmark residues in FR2 and/or FR4 (again, including those at positions 108, 103 and/or 45).

As will be clear from the disclosure herein, it is also within the scope of the invention to use natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs (herein collectively referred to as “analogs”) of the Nanobodies of the invention as defined herein, and in particular analogs of the Nanobodies of SEQ ID NO's 868 to 973 and/or 1044 to 1053. Thus, according to one aspect of the invention, the term “Nanobody of the invention” in its broadest sense also covers such analogs.

Generally, in such analogs, one or more amino acid residues may have been replaced, deleted and/or added, compared to the Nanobodies of the invention as defined herein. Such substitutions, insertions or deletions may be made in one or more of the framework regions and/or in one or more of the CDR's. When such substitutions, insertions or deletions are made in one or more of the framework regions, they may be made at one or more of the Hallmark residues and/or at one or more of the other positions in the framework residues, although substitutions, insertions or deletions at the Hallmark residues are generally less preferred (unless these are suitable humanizing substitutions as described herein).

By means of non-limiting examples, a substitution may for example be a conservative substitution (as described herein) and/or an amino acid residue may be replaced by another amino acid residue that naturally occurs at the same position in another V_(HH) domain (see Tables A-5 to A-8 for some non-limiting examples of such substitutions), although the invention is generally not limited thereto. Thus, any one or more substitutions, deletions or insertions, or any combination thereof, that either improve the properties of the Nanobody of the invention or that at least do not detract too much from the desired properties or from the balance or combination of desired properties of the Nanobody of the invention (i.e. to the extent that the Nanobody is no longer suited for its intended use) are included within the scope of the invention. A skilled person will generally be able to determine and select suitable substitutions, deletions or insertions, or suitable combinations of thereof, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible substitutions and determining their influence on the properties of the Nanobodies thus obtained.

For example, and depending on the host organism used to express the Nanobody or polypeptide of the invention, such deletions and/or substitutions may be designed in such a way that one or more sites for post-translational modification (such as one or more glycosylation sites) are removed, as will be within the ability of the person skilled in the art. Alternatively, substitutions or insertions may be designed so as to introduce one or more sites for attachment of functional groups (as described herein), for example to allow site-specific pegylation (again as described herein).

As can be seen from the data on the V_(HH) entropy and V_(HH) variability given in Tables A-5 to A-8 above, some amino acid residues in the framework regions are more conserved than others. Generally, although the invention in its broadest sense is not limited thereto, any substitutions, deletions or insertions are preferably made at positions that are less conserved. Also, generally, amino acid substitutions are preferred over amino acid deletions or insertions.

The analogs are preferably such that they can bind to a metalloproteinase from the ADAM family with an affinity (suitably measured and/or expressed as a K_(D) value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein for the Nanobodies of the invention.

The analogs are preferably also such that they retain the favourable properties the Nanobodies, as described herein.

Also, according to one preferred aspect, the analogs have a degree of sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, such as at least 95% or 99% or more; and/or preferably have at most 20, preferably at most 10, even more preferably at most 5, such as 4, 3, 2 or only 1 amino acid difference (as defined herein), with one of the Nanobodies of SEQ ID NOs: 868 to 973 and/or 1044 to 1053.

Also, the framework sequences and CDR's of the analogs are preferably such that they are in accordance with the preferred aspects defined herein. More generally, as described herein, the analogs will have (a) a Q at position 108; and/or (b) a charged amino acid or a cysteine residue at position 45 and preferably an E at position 44, and more preferably E at position 44 and R at position 45; and/or (c) P, R or S at position 103.

One preferred class of analogs of the Nanobodies of the invention comprise Nanobodies that have been humanized (i.e. compared to the sequence of a naturally occurring Nanobody of the invention). As mentioned in the background art cited herein, such humanization generally involves replacing one or more amino acid residues in the sequence of a naturally occurring V_(HH) with the amino acid residues that occur at the same position in a human V_(H) domain, such as a human V_(H)3 domain. Examples of possible humanizing substitutions or combinations of humanizing substitutions will be clear to the skilled person, for example from the Tables herein, from the possible humanizing substitutions mentioned in the background art cited herein, and/or from a comparison between the sequence of a Nanobody and the sequence of a naturally occurring human V_(H) domain.

The humanizing substitutions should be chosen such that the resulting humanized Nanobodies still retain the favourable properties of Nanobodies as defined herein, and more preferably such that they are as described for analogs in the preceding paragraphs. A skilled person will generally be able to determine and select suitable humanizing substitutions or suitable combinations of humanizing substitutions, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible humanizing substitutions and determining their influence on the properties of the Nanobodies thus obtained.

Generally, as a result of humanization, the Nanobodies of the invention may become more “human-like”, while still retaining the favorable properties of the Nanobodies of the invention as described herein. As a result, such humanized Nanobodies may have several advantages, such as a reduced immunogenicity, compared to the corresponding naturally occurring V_(HH) domains. Again, based on the disclosure herein and optionally after a limited degree of routine experimentation, the skilled person will be able to select humanizing substitutions or suitable combinations of humanizing substitutions which optimize or achieve a desired or suitable balance between the favourable properties provided by the humanizing substitutions on the one hand and the favourable properties of naturally occurring V_(HH) domains on the other hand.

The Nanobodies of the invention may be suitably humanized at any framework residue(s), such as at one or more Hallmark residues (as defined herein) or at one or more other framework residues (i.e. non-Hallmark residues) or any suitable combination thereof. One preferred humanizing substitution for Nanobodies of the “P,R,S-103 group” or the “KERE group” is Q108 into L108. Nanobodies of the “GLEW class” may also be humanized by a Q108 into L108 substitution, provided at least one of the other Hallmark residues contains a camelid (camelizing) substitution (as defined herein). For example, as mentioned above, one particularly preferred class of humanized Nanobodies has GLEW or a GLEW-like sequence at positions 44-47; P, R or S (and in particular R) at position 103, and an L at position 108.

The humanized and other analogs, and nucleic acid sequences encoding the same, can be provided in any manner known per se. For example, the analogs can be obtained by providing a nucleic acid that encodes a naturally occurring V_(HH) domain, changing the codons for the one or more amino acid residues that are to be substituted into the codons for the corresponding desired amino acid residues (e.g. by site-directed mutagenesis or by PCR using suitable mismatch primers), expressing the nucleic acid/nucleotide sequence thus obtained in a suitable host or expression system; and optionally isolating and/or purifying the analog thus obtained to provide said analog in essentially isolated form (e.g. as further described herein). This can generally be performed using methods and techniques known per se, which will be clear to the skilled person, for example from the handbooks and references cited herein, the background art cited herein and/or from the further description herein. Alternatively, a nucleic acid encoding the desired analog can be synthesized in a manner known per se (for example using an automated apparatus for synthesizing nucleic acid sequences with a predefined amino acid sequence) and can then be expressed as described herein. Yet another technique may involve combining one or more naturally occurring and/or synthetic nucleic acid sequences each encoding a part of the desired analog, and then expressing the combined nucleic acid sequence as described herein. Also, the analogs can be provided using chemical synthesis of the pertinent amino acid sequence using techniques for peptide synthesis known per se, such as those mentioned herein.

In this respect, it will be also be clear to the skilled person that the Nanobodies of the invention (including their analogs) can be designed and/or prepared starting from human V_(H) sequences (i.e. amino acid sequences or the corresponding nucleotide sequences), such as for example from human V_(H)3 sequences such as DP-47, DP-51 or DP-29, i.e. by introducing one or more camelizing substitutions (i.e. changing one or more amino acid residues in the amino acid sequence of said human V_(H) domain into the amino acid residues that occur at the corresponding position in a V_(HH) domain), so as to provide the sequence of a Nanobody of the invention and/or so as to confer the favourable properties of a Nanobody to the sequence thus obtained. Again, this can generally be performed using the various methods and techniques referred to in the previous paragraph, using an amino acid sequence and/or nucleotide sequence for a human V_(H) domain as a starting point.

Some preferred, but non-limiting camelizing substitutions can be derived from Tables A-5-A-8. It will also be clear that camelizing substitutions at one or more of the Hallmark residues will generally have a greater influence on the desired properties than substitutions at one or more of the other amino acid positions, although both and any suitable combination thereof are included within the scope of the invention. For example, it is possible to introduce one or more camelizing substitutions that already confer at least some the desired properties, and then to introduce further camelizing substitutions that either further improve said properties and/or confer additional favourable properties. Again, the skilled person will generally be able to determine and select suitable camelizing substitutions or suitable combinations of camelizing substitutions, based on the disclosure herein and optionally after a limited degree of routine experimentation, which may for example involve introducing a limited number of possible camelizing substitutions and determining whether the favourable properties of Nanobodies are obtained or improved (i.e. compared to the original V_(H) domain). Generally, however, such camelizing substitutions are preferably such that the resulting an amino acid sequence at least contains (a) a Q at position 108; and/or (b) a charged amino acid or a cysteine residue at position 45 and preferably also an E at position 44, and more preferably E at position 44 and R at position 45; and/or (c) P, R or S at position 103; and optionally one or more further camelizing substitutions. More preferably, the camelizing substitutions are such that they result in a Nanobody of the invention and/or in an analog thereof (as defined herein), such as in a humanized analog and/or preferably in an analog that is as defined in the preceding paragraphs.

As will also be clear from the disclosure herein, it is also within the scope of the invention to use parts or fragments, or combinations of two or more parts or fragments, of the Nanobodies of the invention as defined herein, and in particular parts or fragments of the Nanobodies of SEQ ID NO's: 868 to 973 and/or 1044 to 1053. Thus, according to one aspect of the invention, the term “Nanobody of the invention” in its broadest sense also covers such parts or fragments.

Generally, such parts or fragments of the Nanobodies of the invention (including analogs thereof) have amino acid sequences in which, compared to the amino acid sequence of the corresponding full length Nanobody of the invention (or analog thereof), one or more of the amino acid residues at the N-terminal end, one or more amino acid residues at the C-terminal end, one or more contiguous internal amino acid residues, or any combination thereof, have been deleted and/or removed.

The parts or fragments are preferably such that they can bind to a metalloproteinase from the ADAM family with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein for the Nanobodies of the invention.

Any part or fragment is preferably such that it comprises at least 10 contiguous amino acid residues, preferably at least 20 contiguous amino acid residues, more preferably at least 30 contiguous amino acid residues, such as at least 40 contiguous amino acid residues, of the amino acid sequence of the corresponding full length Nanobody of the invention.

Also, any part or fragment is such preferably that it comprises at least one of CDR1, CDR2 and/or CDR3 or at least part thereof (and in particular at least CDR3 or at least part thereof). More preferably, any part or fragment is such that it comprises at least one of the CDR's (and preferably at least CDR3 or part thereof) and at least one other CDR (i.e. CDR1 or CDR2) or at least part thereof, preferably connected by suitable framework sequence(s) or at least part thereof. More preferably, any part or fragment is such that it comprises at least one of the CDR's (and preferably at least CDR3 or part thereof) and at least part of the two remaining CDR's, again preferably connected by suitable framework sequence(s) or at least part thereof.

According to another particularly preferred, but non-limiting aspect, such a part or fragment comprises at least CDR3, such as FR3, CDR3 and FR4 of the corresponding full length Nanobody of the invention, i.e. as for example described in the International application WO 03/050531 (Lasters et al.).

As already mentioned above, it is also possible to combine two or more of such parts or fragments (i.e. from the same or different Nanobodies of the invention), i.e. to provide an analog (as defined herein) and/or to provide further parts or fragments (as defined herein) of a Nanobody of the invention. It is for example also possible to combine one or more parts or fragments of a Nanobody of the invention with one or more parts or fragments of a human V_(H) domain.

According to one preferred aspect, the parts or fragments have a degree of sequence identity of at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, such as at least 90%, 95% or 99% or more with one of the Nanobodies of SEQ ID NOs 868 to 973 and/or 1044 to 1053.

The parts and fragments, and nucleic acid sequences encoding the same, can be provided and optionally combined in any manner known per se. For example, such parts or fragments can be obtained by inserting a stop codon in a nucleic acid that encodes a full-sized Nanobody of the invention, and then expressing the nucleic acid thus obtained in a manner known per se (e.g. as described herein). Alternatively, nucleic acids encoding such parts or fragments can be obtained by suitably restricting a nucleic acid that encodes a full-sized Nanobody of the invention or by synthesizing such a nucleic acid in a manner known per se. Parts or fragments may also be provided using techniques for peptide synthesis known per se.

The invention in its broadest sense also comprises derivatives of the Nanobodies of the invention. Such derivatives can generally be obtained by modification, and in particular by chemical and/or biological (e.g. enzymatical) modification, of the Nanobodies of the invention and/or of one or more of the amino acid residues that form the Nanobodies of the invention.

Examples of such modifications, as well as examples of amino acid residues within the Nanobody sequence that can be modified in such a manner (i.e. either on the protein backbone but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such modifications will be clear to the skilled person.

For example, such a modification may involve the introduction (e.g. by covalent linking or in an other suitable manner) of one or more functional groups, residues or moieties into or onto the Nanobody of the invention, and in particular of one or more functional groups, residues or moieties that confer one or more desired properties or functionalities to the Nanobody of the invention. Example of such functional groups will be clear to the skilled person.

For example, such modification may comprise the introduction (e.g. by covalent binding or in any other suitable manner) of one or more functional groups that increase the half-life, the solubility and/or the absorption of the Nanobody of the invention, that reduce the immunogenicity and/or the toxicity of the Nanobody of the invention, that eliminate or attenuate any undesirable side effects of the Nanobody of the invention, and/or that confer other advantageous properties to and/or reduce the undesired properties of the Nanobodies and/or polypeptides of the invention; or any combination of two or more of the foregoing. Examples of such functional groups and of techniques for introducing them will be clear to the skilled person, and can generally comprise all functional groups and techniques mentioned in the general background art cited hereinabove as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody fragments (including ScFv's and single domain antibodies), for which reference is for example made to Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Pa. (1980). Such functional groups may for example be linked directly (for example covalently) to a Nanobody of the invention, or optionally via a suitable linker or spacer, as will again be clear to the skilled person.

One of the most widely used techniques for increasing the half-life and/or reducing the immunogenicity of pharmaceutical proteins comprises attachment of a suitable pharmacologically acceptable polymer, such as poly(ethyleneglycol) (PEG) or derivatives thereof (such as methoxypoly(ethyleneglycol) or mPEG). Generally, any suitable form of pegylation can be used, such as the pegylation used in the art for antibodies and antibody fragments (including but not limited to (single) domain antibodies and ScFv's); reference is made to for example Chapman, Nat. Biotechnol., 54, 531-545 (2002); by Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), by Harris and Chess, Nat. Rev. Drug. Discov., 2, (2003) and in WO 04/060965. Various reagents for pegylation of proteins are also commercially available, for example from Nektar Therapeutics, USA.

Preferably, site-directed pegylation is used, in particular via a cysteine-residue (see for example Yang et al., Protein Engineering, 16, 10, 761-770 (2003). For example, for this purpose, PEG may be attached to a cysteine residue that naturally occurs in a Nanobody of the invention, a Nanobody of the invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus of a Nanobody of the invention, all using techniques of protein engineering known per se to the skilled person.

Preferably, for the Nanobodies and proteins of the invention, a PEG is used with a molecular weight of more than 5000, such as more than 10,000 and less than 200,000, such as less than 100,000; for example in the range of 20,000-80,000.

Another, usually less preferred modification comprises N-linked or O-linked glycosylation, usually as part of co-translational and/or post-translational modification, depending on the host cell used for expressing the Nanobody or polypeptide of the invention.

Yet another modification may comprise the introduction of one or more detectable labels or other signal-generating groups or moieties, depending on the intended use of the labelled Nanobody. Suitable labels and techniques for attaching, using and detecting them will be clear to the skilled person, and for example include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent metals such as ¹⁵²Eu or others metals from the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs), radio-isotopes (such as ³H, ¹²⁵I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, and ⁷⁵Se), metals, metal chelates or metallic cations (for example metallic cations such as ^(99m)Tc, ¹²³I, ¹¹¹In, ¹³¹I, ⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, and ⁶⁸Ga or other metals or metallic cations that are particularly suited for use in in vivo, in vitro or in situ diagnosis and imaging, such as (¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr, and ⁵⁶Fe), as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase). Other suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy.

Such labelled Nanobodies and polypeptides of the invention may for example be used for in vitro, in vivo or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA and other “sandwich assays”, etc.) as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label.

As will be clear to the skilled person, another modification may involve the introduction of a chelating group, for example to chelate one of the metals or metallic cations referred to above. Suitable chelating groups for example include, without limitation, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

Yet another modification may comprise the introduction of a functional group that is one part of a specific binding pair, such as the biotin-(strept)avidin binding pair. Such a functional group may be used to link the Nanobody of the invention to another protein, polypeptide or chemical compound that is bound to the other half of the binding pair, i.e. through formation of the binding pair. For example, a Nanobody of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such a conjugated Nanobody may be used as a reporter, for example in a diagnostic system where a detectable signal-producing agent is conjugated to avidin or streptavidin. Such binding pairs may for example also be used to bind the Nanobody of the invention to a carrier, including carriers suitable for pharmaceutical purposes. One non-limiting example are the liposomal formulations described by Cao and Suresh, Journal of Drug Targeting, 8, 4, 257 (2000). Such binding pairs may also be used to link a therapeutically active agent to the Nanobody of the invention.

For some applications, in particular for those applications in which it is intended to kill a cell that expresses the target against which the Nanobodies of the invention are directed (e.g. in the treatment of cancer), or to reduce or slow the growth and/or proliferation such a cell, the Nanobodies of the invention may also be linked to a toxin or to a toxic residue or moiety. Examples of toxic moieties, compounds or residues which can be linked to a Nanobody of the invention to provide—for example—a cytotoxic compound will be clear to the skilled person and can for example be found in the prior art cited above and/or in the further description herein. One example is the so-called ADEPT™ technology described in WO 03/055527.

Other potential chemical and enzymatical modifications will be clear to the skilled person. Such modifications may also be introduced for research purposes (e.g. to study function-activity relationships). Reference is for example made to Lundblad and Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).

Preferably, the derivatives are such that they bind to a metalloproteinase from the ADAM family with an affinity (suitably measured and/or expressed as a K_(D)-value (actual or apparent), a K_(A)-value (actual or apparent), a k_(on)-rate and/or a k_(off)-rate, or alternatively as an IC₅₀ value, as further described herein) that is as defined herein for the Nanobodies of the invention.

As mentioned above, the invention also relates to proteins or polypeptides that essentially consist of or comprise at least one Nanobody of the invention. By “essentially consist of” is meant that the amino acid sequence of the polypeptide of the invention either is exactly the same as the amino acid sequence of a Nanobody of the invention or corresponds to the amino acid sequence of a Nanobody of the invention which has a limited number of amino acid residues, such as 1-20 amino acid residues, for example 1-10 amino acid residues and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, added at the amino terminal end, at the carboxy terminal end, or at both the amino terminal end and the carboxy terminal end of the amino acid sequence of the Nanobody.

Said amino acid residues may or may not change, alter or otherwise influence the (biological) properties of the Nanobody and may or may not add further functionality to the Nanobody. For example, such amino acid residues:

-   -   can comprise an N-terminal Met residue, for example as result of         expression in a heterologous host cell or host organism.     -   may form a signal sequence or leader sequence that directs         secretion of the Nanobody from a host cell upon synthesis.         Suitable secretory leader peptides will be clear to the skilled         person, and may be as further described herein. Usually, such a         leader sequence will be linked to the N-terminus of the         Nanobody, although the invention in its broadest sense is not         limited thereto;     -   may form a sequence or signal that allows the Nanobody to be         directed towards and/or to penetrate or enter into specific         organs, tissues, cells, or parts or compartments of cells,         and/or that allows the Nanobody to penetrate or cross a         biological barrier such as a cell membrane, a cell layer such as         a layer of epithelial cells, a tumor including solid tumors, or         the blood-brain-barrier. Examples of such amino acid sequences         will be clear to the skilled person. Some non-limiting examples         are the small peptide vectors (“Pep-trans vectors”) described in         WO 03/026700 and in Temsamani et al., Expert Opin. Biol. Ther.,         1, 773 (2001); Temsamani and Vidal, Drug Discov. Today, 9,         1012 (004) and Rousselle, J. Pharmacol. Exp. Ther., 296, 124-131         (2001), and the membrane translocator sequence described by Zhao         et al., Apoptosis, 8, 631-637 (2003). C-terminal and N-terminal         amino acid sequences for intracellular targeting of antibody         fragments are for example described by Cardinale et al.,         Methods, 34, 171 (2004). Other suitable techniques for         intracellular targeting involve the expression and/or use of         so-called “intrabodies” comprising a Nanobody of the invention,         as mentioned below;     -   may form a “tag”, for example an amino acid sequence or residue         that allows or facilitates the purification of the Nanobody, for         example using affinity techniques directed against said sequence         or residue. Thereafter, said sequence or residue may be removed         (e.g. by chemical or enzymatical cleavage) to provide the         Nanobody sequence (for this purpose, the tag may optionally be         linked to the Nanobody sequence via a cleavable linker sequence         or contain a cleavable motif). Some preferred, but non-limiting         examples of such residues are multiple histidine residues,         glutathione residues and a myc-tag (see for example SEQ ID NO:31         of WO 06/12282).     -   may be one or more amino acid residues that have been         functionalized and/or that can serve as a site for attachment of         functional groups. Suitable amino acid residues and functional         groups will be clear to the skilled person and include, but are         not limited to, the amino acid residues and functional groups         mentioned herein for the derivatives of the Nanobodies of the         invention.

According to another aspect, a polypeptide of the invention comprises a Nanobody of the invention, which is fused at its amino terminal end, at its carboxy terminal end, or both at its amino terminal end and at its carboxy terminal end to at least one further amino acid sequence, i.e. so as to provide a fusion protein comprising said Nanobody of the invention and the one or more further amino acid sequences. Such a fusion will also be referred to herein as a “Nanobody fusion”.

The one or more further amino acid sequence may be any suitable and/or desired amino acid sequences. The further amino acid sequences may or may not change, alter or otherwise influence the (biological) properties of the Nanobody, and may or may not add further functionality to the Nanobody or the polypeptide of the invention. Preferably, the further amino acid sequence is such that it confers one or more desired properties or functionalities to the Nanobody or the polypeptide of the invention.

For example, the further amino acid sequence may also provide a second binding site, which binding site may be directed against any desired protein, polypeptide, antigen, antigenic determinant or epitope (including but not limited to the same protein, polypeptide, antigen, antigenic determinant or epitope against which the Nanobody of the invention is directed, or a different protein, polypeptide, antigen, antigenic determinant or epitope).

Example of such amino acid sequences will be clear to the skilled person, and may generally comprise all amino acid sequences that are used in peptide fusions based on conventional antibodies and fragments thereof (including but not limited to ScFv's and single domain antibodies). Reference is for example made to the review by Holliger and Hudson, Nature Biotechnology, 23, 9, 1126-1136 (2005),

For example, such an amino acid sequence may be an amino acid sequence that increases the half-life, the solubility, or the absorption, reduces the immunogenicity or the toxicity, eliminates or attenuates undesirable side effects, and/or confers other advantageous properties to and/or reduces the undesired properties of the polypeptides of the invention, compared to the Nanobody of the invention per se. Some non-limiting examples of such amino acid sequences are serum proteins, such as human serum albumin (see for example WO 00/27435) or haptenic molecules (for example haptens that are recognized by circulating antibodies, see for example WO 98/22141).

In particular, it has been described in the art that linking fragments of immunoglobulins (such as V_(H) domains) to serum albumin or to fragments thereof can be used to increase the half-life. Reference is for made to WO 00/27435 and WO 01/077137). According to the invention, the Nanobody of the invention is preferably either directly linked to serum albumin (or to a suitable fragment thereof) or via a suitable linker, and in particular via a suitable peptide linked so that the polypeptide of the invention can be expressed as a genetic fusion (protein). According to one specific aspect, the Nanobody of the invention may be linked to a fragment of serum albumin that at least comprises the domain III of serum albumin or part thereof. Reference is for example made to the U.S. provisional application 60/788,256 of Ablynx N.V. entitled “Albumin derived amino acid sequence, use thereof for increasing the half-life of therapeutic proteins and of other therapeutic proteins and entities, and constructs comprising the same” filed on Mar. 31, 2006 (see also PCT/EP2007/002817).

Alternatively, the further amino acid sequence may provide a second binding site or binding unit that is directed against a serum protein (such as, for example, human serum albumin or another serum protein such as IgG), so as to provide increased half-life in serum. Such amino acid sequences for example include the Nanobodies described below, as well as the small peptides and binding proteins described in WO 91/01743, WO 01/45746 and WO 02/076489 and the dAb's described in WO 03/002609 and WO 04/003019. Reference is also made to Harmsen et al., Vaccine, 23 (41); 4926-42, 2005, as well as to EP 0 368 684, as well as to the following the U.S. provisional application 60/843,349 (see also PCT/EP2007/059475), 60/850,774 (see also PCT/EP2007/060849), 60/850,775 (see also PCT/EP2007/060850) by Ablynx N.V. mentioned herein US provisional application of Ablynx N.V. entitled “Peptides capable of binding to serum proteins” filed on Dec. 5, 2006 (see also PCT/EP2007/063348).

Such amino acid sequences may in particular be directed against serum albumin (and more in particular human serum albumin) and/or against IgG (and more in particular human IgG). For example, such amino acid sequences may be amino acid sequences that are directed against (human) serum albumin and amino acid sequences that can bind to amino acid residues on (human) serum albumin that are not involved in binding of serum albumin to FcRn (see for example WO 06/0122787) and/or amino acid sequences that are capable of binding to amino acid residues on serum albumin that do not form part of domain III of serum albumin (see again for example WO 06/0122787); amino acid sequences that have or can provide an increased half-life (see for example the U.S. provisional application 60/843,349 by Ablynx N.V. entitled “Serum albumin binding proteins with long half-lives” filed on Sep. 8, 2006; see also PCT/EP2007/059475); amino acid sequences against human serum albumin that are cross-reactive with serum albumin from at least one species of mammal, and in particular with at least one species of primate (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus), reference is again made to the U.S. provisional application 60/843,349 and PCT/EP2007/059475); amino acid sequences that can bind to serum albumin in a pH independent manner (see for example the U.S. provisional application 60/850,774 by Ablynx N.V. entitled “Amino acid sequences that bind to serum proteins in a manner that is essentially independent of the pH, compounds comprising the same, and uses thereof”, filed on Oct. 11, 2006; see also and PCT/EP2007/059475) and/or amino acid sequences that are conditional binders (see for example the U.S. provisional application 60/850,775 by Ablynx N.V. entitled “Amino acid sequences that bind to a desired molecule in a conditional manner”, filed on Oct. 11, 2006; see also PCT/EP2007/060850).

According to another aspect, the one or more further amino acid sequences may comprise one or more parts, fragments or domains of conventional 4-chain antibodies (and in particular human antibodies) and/or of heavy chain antibodies. For example, although usually less preferred, a Nanobody of the invention may be linked to a conventional (preferably human) V_(H) or V_(L) domain or to a natural or synthetic analog of a V_(H) or V_(L) domain, again optionally via a linker sequence (including but not limited to other (single) domain antibodies, such as the dAb's described by Ward et al.).

The at least one Nanobody may also be linked to one or more (preferably human) C_(H)1, C_(H)2 and/or C_(H)3 domains, optionally via a linker sequence. For instance, a Nanobody linked to a suitable C_(H)1 domain could for example be used—together with suitable light chains—to generate antibody fragments/structures analogous to conventional Fab fragments or F(ab′)₂ fragments, but in which one or (in case of an F(ab′)₂ fragment) one or both of the conventional V_(H) domains have been replaced by a Nanobody of the invention. Also, two Nanobodies could be linked to a C_(H)3 domain (optionally via a linker) to provide a construct with increased half-life in vivo.

According to one specific aspect of a polypeptide of the invention, one or more Nanobodies of the invention may be linked (optionally via a suitable linker or hinge region) to one or more constant domains (for example, 2 or 3 constant domains that can be used as part of/to form an Fc portion), to an Fc portion and/or to one or more antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the invention and/or may confer the ability to bind to one or more Fc receptors. For example, for this purpose, and without being limited thereto, the one or more further amino acid sequences may comprise one or more C_(H)2 and/or C_(H)3 domains of an antibody, such as from a heavy chain antibody (as described herein) and more preferably from a conventional human 4-chain antibody; and/or may form (part of) and Fc region, for example from IgG (e.g. from IgG1, IgG2, IgG3 or IgG4), from IgE or from another human Ig such as IgA, IgD or IgM. For example, WO 94/04678 describes heavy chain antibodies comprising a Camelid V_(HH) domain or a humanized derivative thereof (i.e. a Nanobody), in which the Camelidae C_(H)2 and/or C_(H)3 domain have been replaced by human C_(H)2 and C_(H)3 domains, so as to provide an immunoglobulin that consists of 2 heavy chains each comprising a Nanobody and human C_(H)2 and C_(H)3 domains (but no C_(H)1 domain), which immunoglobulin has the effector function provided by the C_(H)2 and C_(H)3 domains and which immunoglobulin can function without the presence of any light chains. Other amino acid sequences that can be suitably linked to the Nanobodies of the invention so as to provide an effector function will be clear to the skilled person, and may be chosen on the basis of the desired effector function(s). Reference is for example made to WO 04/058820, WO 99/42077, WO 02/056910 and WO 05/017148, as well as the review by Holliger and Hudson, supra; and to the non-prepublished US provisional application by Ablynx N.V. entitled “Constructs comprising single variable domains and an Fc portion derived from IgE” which has a filing date of Dec. 4, 2007. Coupling of a Nanobody of the invention to an Fc portion may also lead to an increased half-life, compared to the corresponding Nanobody of the invention. For some applications, the use of an Fc portion and/or of constant domains (i.e. C_(H)2 and/or C_(H)3 domains) that confer increased half-life without any biologically significant effector function may also be suitable or even preferred. Other suitable constructs comprising one or more Nanobodies and one or more constant domains with increased half-life in vivo will be clear to the skilled person, and may for example comprise two Nanobodies linked to a C_(H)3 domain, optionally via a linker sequence. Generally, any fusion protein or derivatives with increased half-life will preferably have a molecular weight of more than 50 kD, the cut-off value for renal absorption.

In another one specific, but non-limiting, aspect, in order to form a polypeptide of the invention, one or more amino acid sequences of the invention may be linked (optionally via a suitable linker or hinge region) to naturally occurring, synthetic or semisynthetic constant domains (or analogs, variants, mutants, parts or fragments thereof) that have a reduced (or essentially no) tendency to self-associate into dimers (i.e. compared to constant domains that naturally occur in conventional 4-chain antibodies). Such monomeric (i.e. not self-associating) Fc chain variants, or fragments thereof, will be clear to the skilled person. For example, Helm et al., J Biol Chem 1996 271 7494, describe monomeric Fcε chain variants that can be used in the polypeptide chains of the invention.

Also, such monomeric Fc chain variants are preferably such that they are still capable of binding to the complement or the relevant Fc receptor(s) (depending on the Fc portion from which they are derived), and/or such that they still have some or all of the effector functions of the Fc portion from which they are derived (or at a reduced level still suitable for the intended use). Alternatively, in such a polypeptide chain of the invention, the monomeric Fc chain may be used to confer increased half-life upon the polypeptide chain, in which case the monomeric Fc chain may also have no or essentially no effector functions.

Bivalent/multivalent, bispecific/multispecific or biparatopic/multiparatopic polypeptides of the invention may also be linked to Fc portions, in order to provide polypeptide constructs of the type that is described in the non-prepublished US provisional application entitled “immunoglobulin constructs” filed on Dec. 4, 2007.

The further amino acid sequences may also form a signal sequence or leader sequence that directs secretion of the Nanobody or the polypeptide of the invention from a host cell upon synthesis (for example to provide a pre-, pro- or prepro-form of the polypeptide of the invention, depending on the host cell used to express the polypeptide of the invention).

The further amino acid sequence may also form a sequence or signal that allows the Nanobody or polypeptide of the invention to be directed towards and/or to penetrate or enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody or polypeptide of the invention to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Suitable examples of such amino acid sequences will be clear to the skilled person, and for example include, but are not limited to, the “Peptrans” vectors mentioned above, the sequences described by Cardinale et al. and the amino acid sequences and antibody fragments known per se that can be used to express or produce the Nanobodies and polypeptides of the invention as so-called “intrabodies”, for example as described in WO 94/02610, WO 95/22618, U.S. Pat. No. 7,004,940, WO 03/014960, WO 99/07414; WO 05/01690; EP 1 512 696; and in Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag; and in Kontermann, Methods 34, (2004), 163-170, and the further references described therein.

For some applications, in particular for those applications in which it is intended to kill a cell that expresses the target against which the Nanobodies of the invention are directed (e.g. in the treatment of cancer), or to reduce or slow the growth and/or proliferation of such a cell, the Nanobodies of the invention may also be linked to a (cyto)toxic protein or polypeptide. Examples of such toxic proteins and polypeptides which can be linked to a Nanobody of the invention to provide—for example—a cytotoxic polypeptide of the invention will be clear to the skilled person and can for example be found in the prior art cited above and/or in the further description herein. One example is the so-called ADEPT™ technology described in WO 03/055527.

According to one preferred, but non-limiting aspect, said one or more further amino acid sequences comprise at least one further Nanobody, so as to provide a polypeptide of the invention that comprises at least two, such as three, four, five or more Nanobodies, in which said Nanobodies may optionally be linked via one or more linker sequences (as defined herein). Polypeptides of the invention that comprise two or more Nanobodies, of which at least one is a Nanobody of the invention, will also be referred to herein as “multivalent” polypeptides of the invention, and the Nanobodies present in such polypeptides will also be referred to herein as being in a “multivalent format”. For example a “bivalent” polypeptide of the invention comprises two Nanobodies, optionally linked via a linker sequence, whereas a “trivalent” polypeptide of the invention comprises three Nanobodies, optionally linked via two linker sequences; etc.; in which at least one of the Nanobodies present in the polypeptide, and up to all of the Nanobodies present in the polypeptide, is/are a Nanobody of the invention.

In a multivalent polypeptide of the invention, the two or more Nanobodies may be the same or different, and may be directed against the same antigen or antigenic determinant (for example against the same part(s) or epitope(s) or against different parts or epitopes) or may alternatively be directed against different antigens or antigenic determinants; or any suitable combination thereof. For example, a bivalent polypeptide of the invention may comprise (a) two identical Nanobodies; (b) a first Nanobody directed against a first antigenic determinant of a protein or antigen and a second Nanobody directed against the same antigenic determinant of said protein or antigen which is different from the first Nanobody; (c) a first Nanobody directed against a first antigenic determinant of a protein or antigen and a second Nanobody directed against another antigenic determinant of said protein or antigen; or (d) a first Nanobody directed against a first protein or antigen and a second Nanobody directed against a second protein or antigen (i.e. different from said first antigen). Similarly, a trivalent polypeptide of the invention may, for example and without being limited thereto. comprise (a) three identical Nanobodies; (b) two identical Nanobody against a first antigenic determinant of an antigen and a third Nanobody directed against a different antigenic determinant of the same antigen; (c) two identical Nanobody against a first antigenic determinant of an antigen and a third Nanobody directed against a second antigen different from said first antigen; (d) a first Nanobody directed against a first antigenic determinant of a first antigen, a second Nanobody directed against a second antigenic determinant of said first antigen and a third Nanobody directed against a second antigen different from said first antigen; or (e) a first Nanobody directed against a first antigen, a second Nanobody directed against a second antigen different from said first antigen, and a third Nanobody directed against a third antigen different from said first and second antigen.

Polypeptides of the invention that contain at least two Nanobodies, in which at least one Nanobody is directed against a first antigen (i.e. against a metalloproteinase from the ADAM family,) and at least one Nanobody is directed against a second antigen (i.e. different from a metalloproteinase from the ADAM family,), will also be referred to as “multispecific” polypeptides of the invention, and the Nanobodies present in such polypeptides will also be referred to herein as being in a “multispecific format”. Thus, for example, a “bispecific” polypeptide of the invention is a polypeptide that comprises at least one Nanobody directed against a first antigen (i.e. a metalloproteinase from the ADAM family,) and at least one further Nanobody directed against a second antigen (i.e. different from a metalloproteinase from the ADAM family,), whereas a “trispecific” polypeptide of the invention is a polypeptide that comprises at least one Nanobody directed against a first antigen (i.e. a metalloproteinase from the ADAM family,), at least one further Nanobody directed against a second antigen (i.e. different from a metalloproteinase from the ADAM family,) and at least one further Nanobody directed against a third antigen (i.e. different from both a metalloproteinase from the ADAM family, and the second antigen); etc.

Accordingly, in its simplest form, a bispecific polypeptide of the invention is a bivalent polypeptide of the invention (as defined herein), comprising a first Nanobody directed against a metalloproteinase from the ADAM family, and a second Nanobody directed against a second antigen, in which said first and second Nanobody may optionally be linked via a linker sequence (as defined herein); whereas a trispecific polypeptide of the invention in its simplest form is a trivalent polypeptide of the invention (as defined herein), comprising a first Nanobody directed against a metalloproteinase from the ADAM family, a second Nanobody directed against a second antigen and a third Nanobody directed against a third antigen, in which said first, second and third Nanobody may optionally be linked via one or more, and in particular one and more, in particular two, linker sequences.

However, as will be clear from the description hereinabove, the invention is not limited thereto, in the sense that a multispecific polypeptide of the invention may comprise at least one Nanobody against a metalloproteinase from the ADAM family, and any number of Nanobodies directed against one or more antigens different from a metalloproteinase from the ADAM family.

Furthermore, although it is encompassed within the scope of the invention that the specific order or arrangement of the various Nanobodies in the polypeptides of the invention may have some influence on the properties of the final polypeptide of the invention (including but not limited to the affinity, specificity or avidity for a metalloproteinase from the ADAM family, or against the one or more other antigens), said order or arrangement is usually not critical and may be suitably chosen by the skilled person, optionally after some limited routine experiments based on the disclosure herein. Thus, when reference is made to a specific multivalent or multispecific polypeptide of the invention, it should be noted that this encompasses any order or arrangements of the relevant Nanobodies, unless explicitly indicated otherwise.

Finally, it is also within the scope of the invention that the polypeptides of the invention contain two or more Nanobodies and one or more further amino acid sequences (as mentioned herein).

For multivalent and multispecific polypeptides containing one or more V_(HH) domains and their preparation, reference is also made to Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001; Muyldermans, Reviews in Molecular Biotechnology 74 (2001), 277-302; as well as to for example WO 96/34103 and WO 99/23221. Some other examples of some specific multispecific and/or multivalent polypeptide of the invention can be found in the applications by Ablynx N.V. referred to herein.

One preferred, but non-limiting example of a multispecific polypeptide of the invention comprises at least one Nanobody of the invention and at least one Nanobody that provides for an increased half-life. Such Nanobodies may for example be Nanobodies that are directed against a serum protein, and in particular a human serum protein, such as human serum albumin, thyroxine-binding protein, (human) transferrin, fibrinogen, an immunoglobulin such as IgG, IgE or IgM, or against one of the serum proteins listed in WO 04/003019. Of these, Nanobodies that can bind to serum albumin (and in particular human serum albumin) or to IgG (and in particular human IgG, see for example Nanobody VH-1 described in the review by Muyldermans, supra) are particularly preferred (although for example, for experiments in mice or primates, Nanobodies against or cross-reactive with mouse serum albumin (MSA) or serum albumin from said primate, respectively, can be used. However, for pharmaceutical use, Nanobodies against human serum albumin or human IgG will usually be preferred). Nanobodies that provide for increased half-life and that can be used in the polypeptides of the invention include the Nanobodies directed against serum albumin that are described in WO 04/041865, in WO 06/122787 and in the further patent applications by Ablynx N.V., such as those mentioned above.

For example, the some preferred Nanobodies that provide for increased half-life for use in the present invention include Nanobodies that can bind to amino acid residues on (human) serum albumin that are not involved in binding of serum albumin to FcRn (see for example WO 06/0122787); Nanobodies that are capable of binding to amino acid residues on serum albumin that do not form part of domain III of serum albumin (see for example WO 06/0122787); Nanobodies that have or can provide an increased half-life (see for example the U.S. provisional application 60/843,349 by Ablynx N.V mentioned herein; see also PCT/EP2007/059475); Nanobodies against human serum albumin that are cross-reactive with serum albumin from at least one species of mammal, and in particular with at least one species of primate (such as, without limitation, monkeys from the genus Macaca (such as, and in particular, cynomolgus monkeys (Macaca fascicularis) and/or rhesus monkeys (Macaca mulatta)) and baboon (Papio ursinus)) (see for example the U.S. provisional application 60/843,349 by Ablynx N.V. see also PCT/EP2007/059475); Nanobodies that can bind to serum albumin in a pH independent manner (see for example the U.S. provisional application 60/850,774 by Ablynx N.V. mentioned herein) and/or Nanobodies that are conditional binders (see for example the U.S. provisional application 60/850,775 by Ablynx N.V.; see also PCT/EP2007/060850).

Some particularly preferred Nanobodies that provide for increased half-life and that can be used in the polypeptides of the invention include the Nanobodies ALB-1 to ALB-10 disclosed in WO 06/122787 (see Tables II and III) of which ALB-8 (SEQ ID NO: 62 in WO 06/122787) is particularly preferred.

According to a specific, but non-limiting aspect of the invention, the polypeptides of the invention contain, besides the one or more Nanobodies of the invention, at least one Nanobody against human serum albumin.

Generally, any polypeptides of the invention with increased half-life that contain one or more Nanobodies of the invention, and any derivatives of Nanobodies of the invention or of such polypeptides that have an increased half-life, preferably have a half-life that is at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding Nanobody of the invention per se. For example, such a derivative or polypeptides with increased half-life may have a half-life that is increased with more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding Nanobody of the invention per se.

In a preferred, but non-limiting aspect of the invention, such derivatives or polypeptides may exhibit a serum half-life in human of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, even more preferably at least 72 hours or more. For example, such derivatives or polypeptides may have a half-life of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days), more preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days (such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days).

According to one aspect of the invention the polypeptides are capable of binding to one or more molecules which can increase the half-life of the polypeptide in vivo.

The polypeptides of the invention are stabilised in vivo and their half-life increased by binding to molecules which resist degradation and/or clearance or sequestration. Typically, such molecules are naturally occurring proteins which themselves have a long half-life in vivo.

Another preferred, but non-limiting example of a multispecific polypeptide of the invention comprises at least one Nanobody of the invention and at least one Nanobody that directs the polypeptide of the invention towards, and/or that allows the polypeptide of the invention to penetrate or to enter into specific organs, tissues, cells, or parts or compartments of cells, and/or that allows the Nanobody to penetrate or cross a biological barrier such as a cell membrane, a cell layer such as a layer of epithelial cells, a tumor including solid tumors, or the blood-brain-barrier. Examples of such Nanobodies include Nanobodies that are directed towards specific cell-surface proteins, markers or epitopes of the desired organ, tissue or cell (for example cell-surface markers associated with tumor cells), and the single-domain brain targeting antibody fragments described in WO 02/057445 and WO 06/040153, of which FC44 (SEQ ID NO: 189 of WO 06/040153) and FC5 (SEQ ID NO: 190 of WO 06/040154) are preferred examples.

In the polypeptides of the invention, the one or more Nanobodies and the one or more polypeptides may be directly linked to each other (as for example described in WO 99/23221) and/or may be linked to each other via one or more suitable spacers or linkers, or any combination thereof.

Suitable spacers or linkers for use in multivalent and multispecific polypeptides will be clear to the skilled person, and may generally be any linker or spacer used in the art to link amino acid sequences. Preferably, said linker or spacer is suitable for use in constructing proteins or polypeptides that are intended for pharmaceutical use.

Some particularly preferred spacers include the spacers and linkers that are used in the art to link antibody fragments or antibody domains. These include the linkers mentioned in the general background art cited above, as well as for example linkers that are used in the art to construct diabodies or ScFv fragments (in this respect, however, its should be noted that, whereas in diabodies and in ScFv fragments, the linker sequence used should have a length, a degree of flexibility and other properties that allow the pertinent V_(H) and V_(L) domains to come together to form the complete antigen-binding site, there is no particular limitation on the length or the flexibility of the linker used in the polypeptide of the invention, since each Nanobody by itself forms a complete antigen-binding site).

For example, a linker may be a suitable amino acid sequence, and in particular amino acid sequences of between 1 and 50, preferably between 1 and 30, such as between 1 and 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, for example of the type (gly_(x)ser_(y))_(z), such as (for example (gly₄ser)₃ or (gly₃ser₂)₃, as described in WO 99/42077 and the GS30, GS15, GS9 and GS7 linkers described in the applications by Ablynx mentioned herein (see for example WO 06/040153 and WO 06/122825), as well as hinge-like regions, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences (such as described in WO 94/04678).

Some other particularly preferred linkers are poly-alanine (such as AAA), as well as the linkers GS30 (SEQ ID NO: 85 in WO 06/122825) and GS9 (SEQ ID NO: 84 in WO 06/122825).

Other suitable linkers generally comprise organic compounds or polymers, in particular those suitable for use in proteins for pharmaceutical use. For instance, poly(ethyleneglycol) moieties have been used to link antibody domains, see for example WO 04/081026.

It is encompassed within the scope of the invention that the length, the degree of flexibility and/or other properties of the linker(s) used (although not critical, as it usually is for linkers used in ScFv fragments) may have some influence on the properties of the final polypeptide of the invention, including but not limited to the affinity, specificity or avidity for a metalloproteinase from the ADAM family, or for one or more of the other antigens. Based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

For example, in multivalent polypeptides of the invention that comprise Nanobodies directed against a multimeric antigen (such as a multimeric receptor or other protein), the length and flexibility of the linker are preferably such that it allows each Nanobody of the invention present in the polypeptide to bind to the antigenic determinant on each of the subunits of the multimer. Similarly, in a multispecific polypeptide of the invention that comprises Nanobodies directed against two or more different antigenic determinants on the same antigen (for example against different epitopes of an antigen and/or against different subunits of a multimeric receptor, channel or protein), the length and flexibility of the linker are preferably such that it allows each Nanobody to bind to its intended antigenic determinant. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

It is also within the scope of the invention that the linker(s) used confer one or more other favourable properties or functionality to the polypeptides of the invention, and/or provide one or more sites for the formation of derivatives and/or for the attachment of functional groups (e.g. as described herein for the derivatives of the Nanobodies of the invention). For example, linkers containing one or more charged amino acid residues (see Table A-2 above) can provide improved hydrophilic properties, whereas linkers that form or contain small epitopes or tags can be used for the purposes of detection, identification and/or purification. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

Finally, when two or more linkers are used in the polypeptides of the invention, these linkers may be the same or different. Again, based on the disclosure herein, the skilled person will be able to determine the optimal linkers for use in a specific polypeptide of the invention, optionally after some limited routine experiments.

Usually, for easy of expression and production, a polypeptide of the invention will be a linear polypeptide. However, the invention in its broadest sense is not limited thereto. For example, when a polypeptide of the invention comprises three of more Nanobodies, it is possible to link them by use of a linker with three or more “arms”, which each “arm” being linked to a Nanobody, so as to provide a “star-shaped” construct. It is also possible, although usually less preferred, to use circular constructs.

The invention also comprises derivatives of the polypeptides of the invention, which may be essentially analogous to the derivatives of the Nanobodies of the invention, i.e. as described herein.

The invention also comprises proteins or polypeptides that “essentially consist” of a polypeptide of the invention (in which the wording “essentially consist of” has essentially the same meaning as indicated hereinabove).

According to one aspect of the invention, the polypeptide of the invention is in essentially isolated from, as defined herein.

The amino acid sequences, Nanobodies, polypeptides and nucleic acids of the invention can be prepared in a manner known per se, as will be clear to the skilled person from the further description herein. For example, the Nanobodies and polypeptides of the invention can be prepared in any manner known per se for the preparation of antibodies and in particular for the preparation of antibody fragments (including but not limited to (single) domain antibodies and ScFv fragments). Some preferred, but non-limiting methods for preparing the amino acid sequences, Nanobodies, polypeptides and nucleic acids include the methods and techniques described herein.

As will be clear to the skilled person, one particularly useful method for preparing an amino acid sequence, Nanobody and/or a polypeptide of the invention generally comprises the steps of:

-   i) the expression, in a suitable host cell or host organism (also     referred to herein as a “host of the invention”) or in another     suitable expression system of a nucleic acid that encodes said amino     acid sequence, Nanobody or polypeptide of the invention (also     referred to herein as a “nucleic acid of the invention”), optionally     followed by: -   ii) isolating and/or purifying the amino acid sequence, Nanobody or     polypeptide of the invention thus obtained.

In particular, such a method may comprise the steps of:

-   i) cultivating and/or maintaining a host of the invention under     conditions that are such that said host of the invention expresses     and/or produces at least one amino acid sequence, Nanobody and/or     polypeptide of the invention; optionally followed by: -   ii) isolating and/or purifying the amino acid sequence, Nanobody or     polypeptide of the invention thus obtained.

A nucleic acid of the invention can be in the form of single or double stranded DNA or RNA, and is preferably in the form of double stranded DNA. For example, the nucleotide sequences of the invention may be genomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage that has been specifically adapted for expression in the intended host cell or host organism).

According to one aspect of the invention, the nucleic acid of the invention is in essentially isolated from, as defined herein.

The nucleic acid of the invention may also be in the form of, be present in and/or be part of a vector, such as for example a plasmid, cosmid or YAC, which again may be in essentially isolated form.

The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information on the amino acid sequences for the polypeptides of the invention given herein, and/or can be isolated from a suitable natural source. To provide analogs, nucleotide sequences encoding naturally occurring V_(HH) domains can for example be subjected to site-directed mutagenesis, so at to provide a nucleic acid of the invention encoding said analog. Also, as will be clear to the skilled person, to prepare a nucleic acid of the invention, also several nucleotide sequences, such as at least one nucleotide sequence encoding a Nanobody and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner.

Techniques for generating the nucleic acids of the invention will be clear to the skilled person and may for instance include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and/or regions that may easily be digested and/or ligated using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more “mismatched” primers, using for example a sequence of a naturally occurring form of a metalloproteinase from the ADAM family as a template. These and other techniques will be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et al. and Ausubel et al., mentioned above, as well as the Examples below.

The nucleic acid of the invention may also be in the form of, be present in and/or be part of a genetic construct, as will be clear to the person skilled in the art. Such genetic constructs generally comprise at least one nucleic acid of the invention that is optionally linked to one or more elements of genetic constructs known per se, such as for example one or more suitable regulatory elements (such as a suitable promoter(s), enhancer(s), terminator(s), etc.) and the further elements of genetic constructs referred to herein. Such genetic constructs comprising at least one nucleic acid of the invention will also be referred to herein as “genetic constructs of the invention”.

The genetic constructs of the invention may be DNA or RNA, and are preferably double-stranded DNA. The genetic constructs of the invention may also be in a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell or in a form suitable for independent replication, maintenance and/or inheritance in the intended host organism. For instance, the genetic constructs of the invention may be in the form of a vector, such as for example a plasmid, cosmid, YAC, a viral vector or transposon. In particular, the vector may be an expression vector, i.e. a vector that can provide for expression in vitro and/or in vivo (e.g. in a suitable host cell, host organism and/or expression system).

In a preferred but non-limiting aspect, a genetic construct of the invention comprises

-   i) at least one nucleic acid of the invention; operably connected to -   ii) one or more regulatory elements, such as a promoter and     optionally a suitable terminator;     and optionally also -   iii) one or more further elements of genetic constructs known per     se;     in which the terms “regulatory element”, “promoter”, “terminator”     and “operably connected” have their usual meaning in the art (as     further described herein); and in which said “further elements”     present in the genetic constructs may for example be 3′- or 5′-UTR     sequences, leader sequences, selection markers, expression     markers/reporter genes, and/or elements that may facilitate or     increase (the efficiency of) transformation or integration. These     and other suitable elements for such genetic constructs will be     clear to the skilled person, and may for instance depend upon the     type of construct used, the intended host cell or host organism; the     manner in which the nucleotide sequences of the invention of     interest are to be expressed (e.g. via constitutive, transient or     inducible expression); and/or the transformation technique to be     used. For example, regulatory sequences, promoters and terminators     known per se for the expression and production of antibodies and     antibody fragments (including but not limited to (single) domain     antibodies and ScFv fragments) may be used in an essentially     analogous manner.

Preferably, in the genetic constructs of the invention, said at least one nucleic acid of the invention and said regulatory elements, and optionally said one or more further elements, are “operably linked” to each other, by which is generally meant that they are in a functional relationship with each other. For instance, a promoter is considered “operably linked” to a coding sequence if said promoter is able to initiate or otherwise control/regulate the transcription and/or the expression of a coding sequence (in which said coding sequence should be understood as being “under the control of” said promoter). Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may also not be required.

Preferably, the regulatory and further elements of the genetic constructs of the invention are such that they are capable of providing their intended biological function in the intended host cell or host organism.

For instance, a promoter, enhancer or terminator should be “operable” in the intended host cell or host organism, by which is meant that (for example) said promoter should be capable of initiating or otherwise controlling/regulating the transcription and/or the expression of a nucleotide sequence—e.g. a coding sequence—to which it is operably linked (as defined herein).

Some particularly preferred promoters include, but are not limited to, promoters known per se for the expression in the host cells mentioned herein; and in particular promoters for the expression in the bacterial cells, such as those mentioned herein and/or those used in the Examples.

A selection marker should be such that it allows—i.e. under appropriate selection conditions—host cells and/or host organisms that have been (successfully) transformed with the nucleotide sequence of the invention to be distinguished from host cells/organisms that have not been (successfully) transformed. Some preferred, but non-limiting examples of such markers are genes that provide resistance against antibiotics (such as kanamycin or ampicillin), genes that provide for temperature resistance, or genes that allow the host cell or host organism to be maintained in the absence of certain factors, compounds and/or (food) components in the medium that are essential for survival of the non-transformed cells or organisms.

A leader sequence should be such that—in the intended host cell or host organism—it allows for the desired post-translational modifications and/or such that it directs the transcribed mRNA to a desired part or organelle of a cell. A leader sequence may also allow for secretion of the expression product from said cell. As such, the leader sequence may be any pro-, pre-, or prepro-sequence operable in the host cell or host organism. Leader sequences may not be required for expression in a bacterial cell. For example, leader sequences known per se for the expression and production of antibodies and antibody fragments (including but not limited to single domain antibodies and ScFv fragments) may be used in an essentially analogous manner.

An expression marker or reporter gene should be such that—in the host cell or host organism—it allows for detection of the expression of (a gene or nucleotide sequence present on) the genetic construct. An expression marker may optionally also allow for the localisation of the expressed product, e.g. in a specific part or organelle of a cell and/or in (a) specific cell(s), tissue(s), organ(s) or part(s) of a multicellular organism. Such reporter genes may also be expressed as a protein fusion with the amino acid sequence of the invention. Some preferred, but non-limiting examples include fluorescent proteins such as GFP.

Some preferred, but non-limiting examples of suitable promoters, terminator and further elements include those that can be used for the expression in the host cells mentioned herein; and in particular those that are suitable for expression in bacterial cells, such as those mentioned herein and/or those used in the Examples below. For some (further) non-limiting examples of the promoters, selection markers, leader sequences, expression markers and further elements that may be present/used in the genetic constructs of the invention—such as terminators, transcriptional and/or translational enhancers and/or integration factors—reference is made to the general handbooks such as Sambrook et al. and Ausubel et al. mentioned above, as well as to the examples that are given in WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, U.S. Pat. No. 7,207,410, U.S. Pat. No. 5,693,492 and EP 1 085 089. Other examples will be clear to the skilled person. Reference is also made to the general background art cited above and the further references cited herein.

The genetic constructs of the invention may generally be provided by suitably linking the nucleotide sequence(s) of the invention to the one or more further elements described above, for example using the techniques described in the general handbooks such as Sambrook et al. and Ausubel et al., mentioned above.

Often, the genetic constructs of the invention will be obtained by inserting a nucleotide sequence of the invention in a suitable (expression) vector known per se. Some preferred, but non-limiting examples of suitable expression vectors are those used in the Examples below, as well as those mentioned herein.

The nucleic acids of the invention and/or the genetic constructs of the invention may be used to transform a host cell or host organism, i.e. for expression and/or production of the amino acid sequence, Nanobody or polypeptide of the invention. Suitable hosts or host cells will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism, for example:

-   -   a bacterial strain, including but not limited to gram-negative         strains such as strains of Escherichia coli; of Proteus, for         example of Proteus mirabilis; of Pseudomonas, for example of         Pseudomonas fluorescens; and gram-positive strains such as         strains of Bacillus, for example of Bacillus subtilis or of         Bacillus brevis; of Streptomyces, for example of Streptomyces         lividans; of Staphylococcus, for example of Staphylococcus         carnosus; and of Lactococcus, for example of Lactococcus lactis;     -   a fungal cell, including but not limited to cells from species         of Trichoderma, for example from Trichoderma reesei; of         Neurospora, for example from Neurospora crassa; of Sordaria, for         example from Sordaria macrospora; of Aspergillus, for example         from Aspergillus niger or from Aspergillus sojae; or from other         filamentous fungi;     -   a yeast cell, including but not limited to cells from species of         Saccharomyces, for example of Saccharomyces cerevisiae; of         Schizosaccharomyces, for example of Schizosaccharomyces pombe;         of Pichia, for example of Pichia pastoris or of Pichia         methanolica; of Hansenula, for example of Hansenula polymorpha;         of Kluyveromyces, for example of Kluyveromyces lactis; of         Arxula, for example of Arxula adeninivorans; of Yarrowia, for         example of Yarrowia lipolytica;     -   an amphibian cell or cell line, such as Xenopus oocytes;     -   an insect-derived cell or cell line, such as cells/cell lines         derived from lepidoptera, including but not limited to         Spodoptera SF9 and Sf21 cells or cells/cell lines derived from         Drosophila, such as Schneider and Kc cells;     -   a plant or plant cell, for example in tobacco plants; and/or     -   a mammalian cell or cell line, for example a cell or cell line         derived from a human, a cell or a cell line from mammals         including but not limited to CHO-cells, BHK-cells (for example         BHK-21 cells) and human cells or cell lines such as HeLa, COS         (for example COS-7) and PER.C6 cells;         as well as all other hosts or host cells known per se for the         expression and production of antibodies and antibody fragments         (including but not limited to (single) domain antibodies and         ScFv fragments), which will be clear to the skilled person.         Reference is also made to the general background art cited         hereinabove, as well as to for example WO 94/29457; WO 96/34103;         WO 99/42077; Frenken et al., (1998), supra; Riechmann and         Muyldermans, (1999), supra; van der Linden, (2000), supra;         Thomassen et al., (2002), supra; Joosten et al., (2003), supra;         Joosten et al., (2005), supra; and the further references cited         herein.

The amino acid sequences, Nanobodies and polypeptides of the invention can also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, for example for prophylactic and/or therapeutic purposes (e.g. as a gene therapy). For this purpose, the nucleotide sequences of the invention may be introduced into the cells or tissues in any suitable way, for example as such (e.g. using liposomes) or after they have been inserted into a suitable gene therapy vector (for example derived from retroviruses such as adenovirus, or parvoviruses such as adeno-associated virus). As will also be clear to the skilled person, such gene therapy may be performed in vivo and/or in situ in the body of a patient by administering a nucleic acid of the invention or a suitable gene therapy vector encoding the same to the patient or to specific cells or a specific tissue or organ of the patient; or suitable cells (often taken from the body of the patient to be treated, such as explanted lymphocytes, bone marrow aspirates or tissue biopsies) may be treated in vitro with a nucleotide sequence of the invention and then be suitably (re-)introduced into the body of the patient. All this can be performed using gene therapy vectors, techniques and delivery systems which are well known to the skilled person, and for example described in Culver, K. W., “Gene Therapy”, 1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York, N.Y.); Giordano, Nature F Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Verma, Nature 389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodera, Blood 91; (1998), 30-36; Verma, Gene Ther. 5 (1998), 692-699; Nabel, Ann N.Y. Acad. Sci.: 811 (1997), 289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, U.S. Pat. No. 5,580,859; U.S. Pat. No. 55,895,466; or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640. For example, in situ expression of ScFv fragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and of diabodies (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) has been described in the art.

For expression of the Nanobodies in a cell, they may also be expressed as so-called “intrabodies”, as for example described in WO 94/02610, WO 95/22618 and U.S. Pat. No. 7,004,940; WO 03/014960; in Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag; and in Kontermann, Methods 34, (2004), 163-170.

The amino acid sequences, Nanobodies and polypeptides of the invention can for example also be produced in the milk of transgenic mammals, for example in the milk of rabbits, cows, goats or sheep (see for example U.S. Pat. No. 6,741,957, U.S. Pat. No. 6,304,489 and U.S. Pat. No. 6,849,992 for general techniques for introducing transgenes into mammals), in plants or parts of plants including but not limited to their leaves, flowers, fruits, seed, roots or tubers (for example in tobacco, maize, soybean or alfalfa) or in for example pupae of the silkworm Bombix mori.

Furthermore, the amino acid sequences, Nanobodies and polypeptides of the invention can also be expressed and/or produced in cell-free expression systems, and suitable examples of such systems will be clear to the skilled person. Some preferred, but non-limiting examples include expression in the wheat germ system; in rabbit reticulocyte lysates; or in the E. coli Zubay system.

As mentioned above, one of the advantages of the use of Nanobodies is that the polypeptides based thereon can be prepared through expression in a suitable bacterial system, and suitable bacterial expression systems, vectors, host cells, regulatory elements, etc., will be clear to the skilled person, for example from the references cited above. It should however be noted that the invention in its broadest sense is not limited to expression in bacterial systems.

Preferably, in the invention, an (in vivo or in vitro) expression system, such as a bacterial expression system, is used that provides the polypeptides of the invention in a form that is suitable for pharmaceutical use, and such expression systems will again be clear to the skilled person. As also will be clear to the skilled person, polypeptides of the invention suitable for pharmaceutical use can be prepared using techniques for peptide synthesis.

For production on industrial scale, preferred heterologous hosts for the (industrial) production of Nanobodies or Nanobody-containing protein therapeutics include strains of E. coli, Pichia pastoris, S. cerevisiae that are suitable for large scale expression/production/fermentation, and in particular for large scale pharmaceutical (i.e. GMP grade) expression/production/fermentation. Suitable examples of such strains will be clear to the skilled person. Such strains and production/expression systems are also made available by companies such as Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, in particular Chinese hamster ovary (CHO) cells, can be used for large scale expression/production/fermentation, and in particular for large scale pharmaceutical expression/production/fermentation. Again, such expression/production systems are also made available by some of the companies mentioned above.

The choice of the specific expression system would depend in part on the requirement for certain post-translational modifications, more specifically glycosylation. The production of a Nanobody-containing recombinant protein for which glycosylation is desired or required would necessitate the use of mammalian expression hosts that have the ability to glycosylate the expressed protein. In this respect, it will be clear to the skilled person that the glycosylation pattern obtained (i.e. the kind, number and position of residues attached) will depend on the cell or cell line that is used for the expression. Preferably, either a human cell or cell line is used (i.e. leading to a protein that essentially has a human glycosylation pattern) or another mammalian cell line is used that can provide a glycosylation pattern that is essentially and/or functionally the same as human glycosylation or at least mimics human glycosylation. Generally, prokaryotic hosts such as E. coli do not have the ability to glycosylate proteins, and the use of lower eukaryotes such as yeast usually leads to a glycosylation pattern that differs from human glycosylation. Nevertheless, it should be understood that all the foregoing host cells and expression systems can be used in the invention, depending on the desired amino acid sequence, Nanobody or polypeptide to be obtained.

Thus, according to one non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is glycosylated. According to another non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is non-glycosylated.

According to one preferred, but non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is produced in a bacterial cell, in particular a bacterial cell suitable for large scale pharmaceutical production, such as cells of the strains mentioned above.

According to another preferred, but non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is produced in a yeast cell, in particular a yeast cell suitable for large scale pharmaceutical production, such as cells of the species mentioned above.

According to yet another preferred, but non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is produced in a mammalian cell, in particular in a human cell or in a cell of a human cell line, and more in particular in a human cell or in a cell of a human cell line that is suitable for large scale pharmaceutical production, such as the cell lines mentioned hereinabove.

When expression in a host cell is used to produce the amino acid sequences, Nanobodies and the polypeptides of the invention, the amino acid sequences, Nanobodies and polypeptides of the invention can be produced either intracellullarly (e.g. in the cytosol, in the periplasma or in inclusion bodies) and then isolated from the host cells and optionally further purified; or can be produced extracellularly (e.g. in the medium in which the host cells are cultured) and then isolated from the culture medium and optionally further purified. When eukaryotic host cells are used, extracellular production is usually preferred since this considerably facilitates the further isolation and downstream processing of the Nanobodies and proteins obtained. Bacterial cells such as the strains of E. coli mentioned above normally do not secrete proteins extracellularly, except for a few classes of proteins such as toxins and hemolysin, and secretory production in E. coli refers to the translocation of proteins across the inner membrane to the periplasmic space. Periplasmic production provides several advantages over cytosolic production. For example, the N-terminal amino acid sequence of the secreted product can be identical to the natural gene product after cleavage of the secretion signal sequence by a specific signal peptidase. Also, there appears to be much less protease activity in the periplasm than in the cytoplasm. In addition, protein purification is simpler due to fewer contaminating proteins in the periplasm. Another advantage is that correct disulfide bonds may form because the periplasm provides a more oxidative environment than the cytoplasm. Proteins overexpressed in E. coli are often found in insoluble aggregates, so-called inclusion bodies. These inclusion bodies may be located in the cytosol or in the periplasm; the recovery of biologically active proteins from these inclusion bodies requires a denaturation/refolding process. Many recombinant proteins, including therapeutic proteins, are recovered from inclusion bodies. Alternatively, as will be clear to the skilled person, recombinant strains of bacteria that have been genetically modified so as to secrete a desired protein, and in particular an amino acid sequence, Nanobody or a polypeptide of the invention, can be used.

Thus, according to one non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is an amino acid sequence, Nanobody or polypeptide that has been produced intracellularly and that has been isolated from the host cell, and in particular from a bacterial cell or from an inclusion body in a bacterial cell. According to another non-limiting aspect of the invention, the amino acid sequence, Nanobody or polypeptide of the invention is an amino acid sequence, Nanobody or polypeptide that has been produced extracellularly, and that has been isolated from the medium in which the host cell is cultivated.

Some preferred, but non-limiting promoters for use with these host cells include,

-   -   for expression in E. coli: lac promoter (and derivatives thereof         such as the lacUV5 promoter); arabinose promoter; left-(PL) and         rightward (PR) promoter of phage lambda; promoter of the trp         operon; hybrid lac/trp promoters (tac and trc); T7-promoter         (more specifically that of T7-phage gene 10) and other T-phage         promoters; promoter of the Tn10 tetracycline resistance gene;         engineered variants of the above promoters that include one or         more copies of an extraneous regulatory operator sequence;     -   for expression in S. cerevisiae: constitutive: ADH1 (alcohol         dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c iso-1),         GAPDH (glyceraldehydes-3-phosphate dehydrogenase), PGK1         (phosphoglycerate kinase), PYK1 (pyruvate kinase); regulated:         GAL1,10,7 (galactose metabolic enzymes), ADH2 (alcohol         dehydrogenase 2), PHO5 (acid phosphatase), CUP1 (copper         metallothionein); heterologous: CaMV (cauliflower mosaic virus         35S promoter);     -   for expression in Pichia pastoris: the AOX1 promoter (alcohol         oxidase I);     -   for expression in mammalian cells: human cytomegalovirus (hCMV)         immediate early enhancer/promoter; human cytomegalovirus (hCMV)         immediate early promoter variant that contains two tetracycline         operator sequences such that the promoter can be regulated by         the Tet repressor; Herpes Simplex Virus thymidine kinase (TK)         promoter; Rous Sarcoma Virus long terminal repeat (RSV LTR)         enhancer/promoter; elongation factor 1α (hEF-1α) promoter from         human, chimpanzee, mouse or rat; the SV40 early promoter; HIV-1         long terminal repeat promoter; β-actin promoter;

Some preferred, but non-limiting vectors for use with these host cells include:

-   -   vectors for expression in mammalian cells: pMAMneo (Clontech),         pcDNA3 (Invitrogen), pMC1neo (Stratagene), pSG5 (Stratagene),         EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110),         pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC37199), pRSVneo         (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and         1ZD35 (ATCC 37565), as well as viral-based expression systems,         such as those based on adenovirus;     -   vectors for expression in bacterial cells: pET vectors (Novagen)         and pQE vectors (Qiagen);     -   vectors for expression in yeast or other fungal cells: pYES2         (Invitrogen) and Pichia expression vectors (Invitrogen);     -   vectors for expression in insect cells: pBlueBacII (Invitrogen)         and other baculovirus vectors     -   vectors for expression in plants or plant cells: for example         vectors based on cauliflower mosaic virus or tobacco mosaic         virus, suitable strains of Agrobacterium, or Ti-plasmid based         vectors.

Some preferred, but non-limiting secretory sequences for use with these host cells include:

-   -   for use in bacterial cells such as E. coli: PelB, Bla, OmpA,         OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lpp, LamB, and the         like; TAT signal peptide, hemolysin C-terminal secretion signal;     -   for use in yeast: α-mating factor prepro-sequence, phosphatase         (pho1), invertase (Suc), etc.;     -   for use in mammalian cells: indigenous signal in case the target         protein is of eukaryotic origin; murine Ig κ-chain V-J2-C signal         peptide; etc.

Suitable techniques for transforming a host or host cell of the invention will be clear to the skilled person and may depend on the intended host cell/host organism and the genetic construct to be used. Reference is again made to the handbooks and patent applications mentioned above.

After transformation, a step for detecting and selecting those host cells or host organisms that have been successfully transformed with the nucleotide sequence/genetic construct of the invention may be performed. This may for instance be a selection step based on a selectable marker present in the genetic construct of the invention or a step involving the detection of the amino acid sequence of the invention, e.g. using specific antibodies.

The transformed host cell (which may be in the form or a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) form further aspects of the present invention.

Preferably, these host cells or host organisms are such that they express, or are (at least) capable of expressing (e.g. under suitable conditions), an amino acid sequence, Nanobody or polypeptide of the invention (and in case of a host organism: in at least one cell, part, tissue or organ thereof). The invention also includes further generations, progeny and/or offspring of the host cell or host organism of the invention, that may for instance be obtained by cell division or by sexual or asexual reproduction.

To produce/obtain expression of the amino acid sequences of the invention, the transformed host cell or transformed host organism may generally be kept, maintained and/or cultured under conditions such that the (desired) amino acid sequence, Nanobody or polypeptide of the invention is expressed/produced. Suitable conditions will be clear to the skilled person and will usually depend upon the host cell/host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequence of the invention. Again, reference is made to the handbooks and patent applications mentioned above in the paragraphs on the genetic constructs of the invention.

Generally, suitable conditions may include the use of a suitable medium, the presence of a suitable source of food and/or suitable nutrients, the use of a suitable temperature, and optionally the presence of a suitable inducing factor or compound (e.g. when the nucleotide sequences of the invention are under the control of an inducible promoter); all of which may be selected by the skilled person. Again, under such conditions, the amino acid sequences of the invention may be expressed in a constitutive manner, in a transient manner, or only when suitably induced.

It will also be clear to the skilled person that the amino acid sequence, Nanobody or polypeptide of the invention may (first) be generated in an immature form (as mentioned above), which may then be subjected to post-translational modification, depending on the host cell/host organism used. Also, the amino acid sequence, Nanobody or polypeptide of the invention may be glycosylated, again depending on the host cell/host organism used.

The amino acid sequence, Nanobody or polypeptide of the invention may then be isolated from the host cell/host organism and/or from the medium in which said host cell or host organism was cultivated, using protein isolation and/or purification techniques known per se, such as (preparative) chromatography and/or electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g. using a specific, cleavable amino acid sequence fused with the amino acid sequence, Nanobody or polypeptide of the invention) and/or preparative immunological techniques (i.e. using antibodies against the amino acid sequence to be isolated).

Generally, for pharmaceutical use, the polypeptides of the invention may be formulated as a pharmaceutical preparation or compositions comprising at least one polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more further pharmaceutically active polypeptides and/or compounds. By means of non-limiting examples, such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc. Such suitable administration forms—which may be solid, semi-solid or liquid, depending on the manner of administration—as well as methods and carriers for use in the preparation thereof, will be clear to the skilled person, and are further described herein.

Thus, in a further aspect, the invention relates to a pharmaceutical composition that contains at least one amino acid of the invention, at least one Nanobody of the invention or at least one polypeptide of the invention and at least one suitable carrier, diluent or excipient (i.e. suitable for pharmaceutical use), and optionally one or more further active substances.

Generally, the amino acid sequences, Nanobodies and polypeptides of the invention can be formulated and administered in any suitable manner known per se, for which reference is for example made to the general background art cited above (and in particular to WO 04/041862, WO 04/041863, WO 04/041865 and WO 04/041867) as well as to the standard handbooks, such as Remington's Pharmaceutical Sciences, 18^(th) Ed., Mack Publishing Company, USA (1990) or Remington, the Science and Practice of Pharmacy, 21st Edition, Lippincott Williams and Wilkins (2005).

For example, the amino acid sequences, Nanobodies and polypeptides of the invention may be formulated and administered in any manner known per se for conventional antibodies and antibody fragments (including ScFv's and diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing the same will be clear to the skilled person, and for example include preparations suitable for parenteral administration (for example intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intra-arterial or intrathecal administration) or for topical (i.e. transdermal or intradermal) administration.

Preparations for parenteral administration may for example be sterile solutions, suspensions, dispersions or emulsions that are suitable for infusion or injection. Suitable carriers or diluents for such preparations for example include, without limitation, sterile water and aqueous buffers and solutions such as physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution; water oils; glycerol; ethanol; glycols such as propylene glycol or as well as mineral oils, animal oils and vegetable oils, for example peanut oil, soybean oil, as well as suitable mixtures thereof. Usually, aqueous solutions or suspensions will be preferred.

The amino acid sequences, Nanobodies and polypeptides of the invention can also be administered using gene therapy methods of delivery. See, e.g., U.S. Pat. No. 5,399,346, which is incorporated by reference in its entirety. Using a gene therapy method of delivery, primary cells transfected with the gene encoding an amino acid sequence, Nanobody or polypeptide of the invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, tumors, or cells and can additionally be transfected with signal and stabilization sequences for subcellularly localized expression.

Thus, the amino acid sequences, Nanobodies and polypeptides of the invention may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the amino acid sequences, Nanobodies and polypeptides of the invention may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of the amino acid sequence, Nanobody or polypeptide of the invention. Their percentage in the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of the amino acid sequence, Nanobody or polypeptide of the invention in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the amino acid sequences, Nanobodies and polypeptides of the invention, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the amino acid sequences, Nanobodies and polypeptides of the invention may be incorporated into sustained-release preparations and devices.

Preparations and formulations for oral administration may also be provided with an enteric coating that will allow the constructs of the invention to resist the gastric environment and pass into the intestines. More generally, preparations and formulations for oral administration may be suitably formulated for delivery into any desired part of the gastrointestinal tract. In addition, suitable suppositories may be used for delivery into the gastrointestinal tract.

The amino acid sequences, Nanobodies and polypeptides of the invention may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the amino acid sequences, Nanobodies and polypeptides of the invention or their salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the amino acid sequences, Nanobodies and polypeptides of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

For topical administration, the amino acid sequences, Nanobodies and polypeptides of the invention may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, hydroxyalkyls or glycols or water-alcohol/glycol blends, in which the amino acid sequences, Nanobodies and polypeptides of the invention can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

Examples of useful dermatological compositions which can be used to deliver the amino acid sequences, Nanobodies and polypeptides of the invention to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

Useful dosages of the amino acid sequences, Nanobodies and polypeptides of the invention can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

Generally, the concentration of the amino acid sequences, Nanobodies and polypeptides of the invention in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%.

The amount of the amino acid sequences, Nanobodies and polypeptides of the invention required for use in treatment will vary not only with the particular amino acid sequence, Nanobody or polypeptide selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the amino acid sequences, Nanobodies and polypeptides of the invention varies depending on the target cell, tumor, tissue, graft, or organ.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

An administration regimen could include long-term, daily treatment. By “long-term” is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. See Remington's Pharmaceutical Sciences (Martin, E. W., ed. 4), Mack Publishing Co., Easton, Pa. The dosage can also be adjusted by the individual physician in the event of any complication.

In another aspect, the invention relates to a method for the prevention and/or treatment of at least one ADAM-related diseases and disorders, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

In the context of the present invention, the term “prevention and/or treatment” not only comprises preventing and/or treating the disease, but also generally comprises preventing the onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms associated therewith and/or preventing a further increase in the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated.

The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of, the diseases and disorders mentioned herein.

The invention relates to a method for the prevention and/or treatment of at least one disease or disorder that is associated with a metalloproteinase from the ADAM family, with its biological or pharmacological activity, and/or with the biological pathways or signaling in which a metalloproteinase from the ADAM family is involved, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same. In particular, the invention relates to a method for the prevention and/or treatment of at least one disease or disorder that can be treated by modulating a metalloproteinase from the ADAM family, its biological or pharmacological activity, and/or the biological pathways or signaling in which a metalloproteinase from the ADAM family is involved, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same. In particular, said pharmaceutically effective amount may be an amount that is sufficient to modulate a metalloproteinase from the ADAM family, its biological or pharmacological activity, and/or the biological pathways or signaling in which a metalloproteinase from the ADAM family is involved; and/or an amount that provides a level of the amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention in the circulation that is sufficient to modulate a metalloproteinase from the ADAM family, its biological or pharmacological activity, and/or the biological pathways or signaling in which a metalloproteinase from the ADAM family is involved.

The invention furthermore relates to a method for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering an amino acid sequence of the invention, a Nanobody of the invention or a polypeptide of the invention to a patient, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

More in particular, the invention relates to a method for the prevention and/or treatment of at least one disease or disorder chosen from the group consisting of the diseases and disorders listed herein, said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

In another aspect, the invention relates to a method for immunotherapy, and in particular for passive immunotherapy, which method comprises administering, to a subject suffering from or at risk of the diseases and disorders mentioned herein, a pharmaceutically active amount of an amino acid sequence of the invention, of a Nanobody of the invention, of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the same.

In the above methods, the amino acid sequences, Nanobodies and/or polypeptides of the invention and/or the compositions comprising the same can be administered in any suitable manner, depending on the specific pharmaceutical formulation or composition to be used. Thus, the amino acid sequences, Nanobodies and/or polypeptides of the invention and/or the compositions comprising the same can for example be administered orally, intraperitoneally (e.g. intravenously, subcutaneously, intramuscularly, or via any other route of administration that circumvents the gastrointestinal tract), intranasally, transdermally, topically, by means of a suppository, by inhalation, again depending on the specific pharmaceutical formulation or composition to be used. The clinician will be able to select a suitable route of administration and a suitable pharmaceutical formulation or composition to be used in such administration, depending on the disease or disorder to be prevented or treated and other factors well known to the clinician.

The amino acid sequences, Nanobodies and/or polypeptides of the invention and/or the compositions comprising the same are administered according to a regime of treatment that is suitable for preventing and/or treating the disease or disorder to be prevented or treated. The clinician will generally be able to determine a suitable treatment regimen, depending on factors such as the disease or disorder to be prevented or treated, the severity of the disease to be treated and/or the severity of the symptoms thereof, the specific amino acid sequence, Nanobody or polypeptide of the invention to be used, the specific route of administration and pharmaceutical formulation or composition to be used, the age, gender, weight, diet, general condition of the patient, and similar factors well known to the clinician.

Generally, the treatment regimen will comprise the administration of one or more amino acid sequences, Nanobodies and/or polypeptides of the invention, or of one or more compositions comprising the same, in one or more pharmaceutically effective amounts or doses. The specific amount(s) or doses to administered can be determined by the clinician, again based on the factors cited above.

Generally, for the prevention and/or treatment of the diseases and disorders mentioned herein and depending on the specific disease or disorder to be treated, the potency of the specific amino acid sequence, Nanobody and polypeptide of the invention to be used, the specific route of administration and the specific pharmaceutical formulation or composition used, the amino acid sequences, Nanobodies and polypeptides of the invention will generally be administered in an amount between 1 gram and 0.01 microgram per kg body weight per day, preferably between 0.1 gram and 0.1 microgram per kg body weight per day, such as about 1, 10, 100 or 1000 microgram per kg body weight per day, either continuously (e.g. by infusion), as a single daily dose or as multiple divided doses during the day. The clinician will generally be able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment. Generally, some guidance on the amounts to be administered can be obtained from the amounts usually administered for comparable conventional antibodies or antibody fragments against the same target administered via essentially the same route, taking into account however differences in affinity/avidity, efficacy, biodistribution, half-life and similar factors well known to the skilled person.

Usually, in the above method, a single amino acid sequence, Nanobody or polypeptide of the invention will be used. It is however within the scope of the invention to use two or more amino acid sequences, Nanobodies and/or polypeptides of the invention in combination.

The Nanobodies, amino acid sequences and polypeptides of the invention may also be used in combination with one or more further pharmaceutically active compounds or principles, i.e. as a combined treatment regimen, which may or may not lead to a synergistic effect. Again, the clinician will be able to select such further compounds or principles, as well as a suitable combined treatment regimen, based on the factors cited above and his expert judgement.

In particular, the amino acid sequences, Nanobodies and polypeptides of the invention may be used in combination with other pharmaceutically active compounds or principles that are or can be used for the prevention and/or treatment of the diseases and disorders cited herein, as a result of which a synergistic effect may or may not be obtained. Examples of such compounds and principles, as well as routes, methods and pharmaceutical formulations or compositions for administering them will be clear to the clinician.

When two or more substances or principles are to be used as part of a combined treatment regimen, they can be administered via the same route of administration or via different routes of administration, at essentially the same time or at different times (e.g. essentially simultaneously, consecutively, or according to an alternating regime). When the substances or principles are to be administered simultaneously via the same route of administration, they may be administered as different pharmaceutical formulations or compositions or part of a combined pharmaceutical formulation or composition, as will be clear to the skilled person.

Also, when two or more active substances or principles are to be used as part of a combined treatment regimen, each of the substances or principles may be administered in the same amount and according to the same regimen as used when the compound or principle is used on its own, and such combined use may or may not lead to a synergistic effect. However, when the combined use of the two or more active substances or principles leads to a synergistic effect, it may also be possible to reduce the amount of one, more or all of the substances or principles to be administered, while still achieving the desired therapeutic action. This may for example be useful for avoiding, limiting or reducing any unwanted side-effects that are associated with the use of one or more of the substances or principles when they are used in their usual amounts, while still obtaining the desired pharmaceutical or therapeutic effect.

The effectiveness of the treatment regimen used according to the invention may be determined and/or followed in any manner known per se for the disease or disorder involved, as will be clear to the clinician. The clinician will also be able, where appropriate and on a case-by-case basis, to change or modify a particular treatment regimen, so as to achieve the desired therapeutic effect, to avoid, limit or reduce unwanted side-effects, and/or to achieve an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand.

Generally, the treatment regimen will be followed until the desired therapeutic effect is achieved and/or for as long as the desired therapeutic effect is to be maintained. Again, this can be determined by the clinician.

In another aspect, the invention relates to the use of an amino acid sequence, Nanobody or polypeptide of the invention in the preparation of a pharmaceutical composition for prevention and/or treatment of at least one ADAM-related diseases and disorders; and/or for use in one or more of the methods of treatment mentioned herein.

The subject to be treated may be any warm-blooded animal, but is in particular a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of, the diseases and disorders mentioned herein.

The invention also relates to the use of an amino acid sequence, Nanobody or polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention and/or treatment of at least one disease or disorder that can be prevented and/or treated by administering an amino acid sequence, Nanobody or polypeptide of the invention to a patient.

More in particular, the invention relates to the use of an amino acid sequence, Nanobody or polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention and/or treatment of ADAM-related diseases and disorders, and in particular for the prevention and treatment of one or more of the diseases and disorders listed herein.

Again, in such a pharmaceutical composition, the one or more amino acid sequences, Nanobodies or polypeptides of the invention may also be suitably combined with one or more other active principles, such as those mentioned herein.

Finally, although the use of the Nanobodies of the invention (as defined herein) and of the polypeptides of the invention is much preferred, it will be clear that on the basis of the description herein, the skilled person will also be able to design and/or generate, in an analogous manner, other amino acid sequences and in particular (single) domain antibodies against a metalloproteinase from the ADAM family, as well as polypeptides comprising such (single) domain antibodies.

For example, it will also be clear to the skilled person that it may be possible to “graft” one or more of the CDR's mentioned above for the Nanobodies of the invention onto such (single) domain antibodies or other protein scaffolds, including but not limited to human scaffolds or non-immunoglobulin scaffolds. Suitable scaffolds and techniques for such CDR grafting will be clear to the skilled person and are well known in the art, see for example U.S. Pat. No. 7,180,370, WO 01/27160, EP 0 605 522, EP 0 460 167, U.S. Pat. No. 7,054,297, Nicaise et al., Protein Science (2004), 13:1882-1891; Ewert et al., Methods, 2004 October; 34(2):184-199; Kettleborough et al., Protein Eng. 1991 October; 4(7): 773-783; O'Brien and Jones, Methods Mol. Biol. 2003: 207: 81-100; Skerra, J. Mol. Recognit. 2000: 13: 167-187, and Saerens et al., J. Mol. Biol. 2005 Sep. 23; 352(3):597-607, and the further references cited therein. For example, techniques known per se for grafting mouse or rat CDR's onto human frameworks and scaffolds can be used in an analogous manner to provide chimeric proteins comprising one or more of the CDR's of the Nanobodies of the invention and one or more human framework regions or sequences.

It should also be noted that, when the Nanobodies of the inventions contain one or more other CDR sequences than the preferred CDR sequences mentioned above, these CDR sequences can be obtained in any manner known per se, for example from Nanobodies (preferred), V_(H) domains from conventional antibodies (and in particular from human antibodies), heavy chain antibodies, conventional 4-chain antibodies (such as conventional human 4-chain antibodies) or other immunoglobulin sequences directed against a metalloproteinase from the ADAM family. Such immunoglobulin sequences directed against a metalloproteinase from the ADAM family can be generated in any manner known per se, as will be clear to the skilled person, i.e. by immunization with a metalloproteinase from the ADAM family or by screening a suitable library of immunoglobulin sequences with a metalloproteinase from the ADAM family, or any suitable combination thereof. Optionally, this may be followed by techniques such as random or site-directed mutagenesis and/or other techniques for affinity maturation known per se. Suitable techniques for generating such immunoglobulin sequences will be clear to the skilled person, and for example include the screening techniques reviewed by Hoogenboom, Nature Biotechnology, 23, 9, 1105-1116 (2005) Other techniques for generating immunoglobulins against a specified target include for example the Nanoclone technology (as for example described in the published US patent application 2006-0211088), so-called SLAM technology (as for example described in the European patent application 0 542 810), the use of transgenic mice expressing human immunoglobulins or the well-known hybridoma techniques (see for example Larrick et al, Biotechnology, Vol. 7, 1989, p. 934). All these techniques can be used to generate immunoglobulins against a metalloproteinase from the ADAM family, and the CDR's of such immunoglobulins can be used in the Nanobodies of the invention, i.e. as outlined above. For example, the sequence of such a CDR can be determined, synthesized and/or isolated, and inserted into the sequence of a Nanobody of the invention (e.g. so as to replace the corresponding native CDR), all using techniques known per se such as those described herein, or Nanobodies of the invention containing such CDR's (or nucleic acids encoding the same) can be synthesized de novo, again using the techniques mentioned herein.

Further uses of the amino acid sequences, Nanobodies, polypeptides, nucleic acids, genetic constructs and hosts and host cells of the invention will be clear to the skilled person based on the disclosure herein. For example, and without limitation, the amino acid sequences of the invention can be linked to a suitable carrier or solid support so as to provide a medium than can be used in a manner known per se to purify a metalloproteinase from the ADAM family from compositions and preparations comprising the same. Derivatives of the amino acid sequences of the invention that comprise a suitable detectable label can also be used as markers to determine (qualitatively or quantitatively) the presence of a metalloproteinase from the ADAM family in a composition or preparation or as a marker to selectively detect the presence of a metalloproteinase from the ADAM family on the surface of a cell or tissue (for example, in combination with suitable cell sorting techniques).

The invention will now be further described by means of the following non-limiting Experimental Part and the non-limiting Figures, in which:

FIG. 1 is a graph showing the effect of purified nanobodies (1 uM, 25 molar excess) on the activity of recombinant ADAM9 (150 ng=40 nM, R&D system).

FIG. 2 is a graph showing the effect of increasing amount of purified nanobodies [2× (16 nM) to 50× (400 nM) excess relative to ADAM17) on the activity of recombinant ADAM17 (40 ng=8 nM, R&D system).

FIG. 3 is a similar graph to FIG. 2, showing the effect of increased Nanobody concentration on ADAM17 activity.

FIG. 4 is a graph showing the effect of purified nanobodies (400 nM, 10 molar excess) on the activity of recombinant ADAM10 (200 ng=40 nM, R&D system).

FIG. 5 is a graph showing Effect of the increasing amount of purified nanobodies (excess relative to ADAM10) on the activity of recombinant ADAM10 (R&D system).

FIG. 6 is a graph showing the effect of 50 molar excess of purified nanobodies on the activity of recombinant ADAM10 (R&D system). On top of each column is indicated the percent residual activity.

EXPERIMENTAL PART Example 1 Immunizations

Two llamas (131 and 132) were immunized according to standard protocols with 6 boosts of R&D Systems Cat #2717-PG and a cocktail of ADAM (recombinant human ADAM8 (amino residues 1-497, R&D Systems, Cat. No. 1031-AD, Lot No. MWP04607A), recombinant human ADAM9 (amino residues 1-697, R&D Systems, Cat. No. 939-AD, Lot No. FQO03607A), recombinant human ADAM10 (amino residues 18-672, Cat. No 936-AD, Lot No: FDW12607A), recombinant human ADAM17/TACE (amino residues 1-671, Cat. No. 930-ADB, Lot No. GDU12607A) and recombinant human ADAMTS5 (amino residues 1-622, Cat. No. 2198-AD, Lot No OLY02607A). Blood was collected from these animals after 7 days after boost 6 and 10 days after boost 6.

Example 2 Library Construction

Peripheral blood mononuclear cells were prepared from blood samples using Ficoll-Hypaque according to the manufacturer's instructions. Next, total RNA extracted was extracted from these cells and used as starting material for RT-PCR to amplify Nanobody encoding gene fragments. These fragments were cloned into phagemid vector pAX50. Phage was prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein) and stored at 4° C. for further use.

Example 3 Selections

Phage libraries 131 and 132 were used for selections on the ADAMs that were used for immunization. The ADAMs were coated individually at 5 ug/ml, 0.5 ug/ml or 0 ug/ml (control) on Nunc Maxisorp ELISA plates. Binding phages were eluted from the coated ADAMs with Trypsine (R1).

Output of both R1 selections were analyzed for enrichment factor (phage present in eluate relative to controls). Based on these parameters the best selections were chosen for further analysis. When only few positive clones were recovered, a second round of selection was done using the same condition as for the first round of selection and trypsine elution (R2).

The Nanobody encoding fragments from R1 and R2 were recloned into the plasmid pAX51 according to standard methods and individual colonies were picked up and grown in 96 deep well plates (1 ml volume) and induced by adding IPTG for Nanobody expression. Periplasmic extracts (volume: ˜80 ul) were prepared according to standard methods (see for example the prior art and applications filed by applicant cited herein).

Example 4 Screening for Binding

In order to determine the binding specificity of the Nanobodies to the ADAMs, the clones were tested in an ELISA assay.

In short, 1 ug/ml of indicated ADAM were immobilized directly on Maxisorp microtiter plates (Nunc). Free binding sites were blocked using 4% Marvel in PBS. Next, 5 ul of periplasmic extract containing Nanobody of the different clones in 100 ul 2% Marvel PBST were allowed to bind to the immobilized antigen. After incubation and a wash step, Nanobody binding was revealed using a mouse-anti-myc secondary antibody, which was after a wash step detected with a HRP-conjugated goat-anti-mouse antibody. Binding specificity was determined based on OD values compared to controls having received no Nanobody (low control, FIG. 1 for R1 and FIG. 2 for R2)

TABLE B-1 Positive clones identify by ELISA for each ADAM after the first round of selection (left table) and second round of selection (right table). R1 R2 LIB 131 LIB 132 TOTAL LIB 131 LIB 132 TOTAL (% pos) (% pos) (% pos) (% pos) (% pos) (% pos) ADAM8 17 (77%)  8 (36%) 25 (57%) ADAM8 ADAM9 12 (55%)  6 (27%) 18 (41%) ADAM9 13 (28%) 13 (28%) ADAM10  6 (27%) 2 (9%)  8 (18%) ADAM10 43 (93%) 24 (52%) 67 (73%) ADAM17 0 (0%) 10 (45%) 10 (23%) ADAM17 43 (93%) 43 (93%) ADAMTS5  6 (27%) 14 (64%) 20 (45%) ADAMTS5 28 (61%) 28 (61%) Are depicted the number of positive identified as well as the representative percentage of positive.

Example 5 Screening for Activity

In order to determine the effect of the Nanobodies on the protease activity, we used an available commercial fluorescent peptide substrate to do an ADAM protease assay (R&D system, cat ES003). Shortly, the periplasms containing the Nanobodies (dialysed against Tris pH9) were preincubated with the ADAM protein for 1 h. Next, the fluorogenic peptide substrate (R&D system, cat ES003) was added and the cleaved product detected in time at 25 C according to manufacturer instruction.

Out of 25 periplasm tested for ADAM17, 6 were inhibiting (less than 80% residual activity) and 6 activating (more than 120% initial activity) compared to other periplasms.

Out of 27 periplasm tested for ADAM10, 5 inhibited significantly (less than 40% residual activity) and 6 activated significantly (more than 200% initial activity) compared to other periplasms.

Out of 31 periplasm tested for ADAM9, 7 showed significant inhibition and 9 were significantly activating.

To confirm the effect of the nanobodies against the ADAMs, the nanobodies were purified, dialysed against 20 nM tris pH9 to avoid the presence of inactivating salts.

The results for the nanobodies against ADAM9 are shown in FIG. 1, which schematically shows the effect of purified nanobodies (1 uM, 25 molar excess) on the activity of recombinant ADAM9 (150 ng=40 nM, R&D system). Because of the somehow stabilizing effect that the presence of non relevant protein has on the ADAM9, the average activity was setup between 110 and 160 fluorescent unit (FU). As can be seen from FIG. 1, nanobodies 30F2, 30D1, 30B6 (also 30E12, but not shown here) have an inactivating effect and Nanobodies 41B5, 30E6, 30C1, 31E12 and 30G12 have an activating effect.

A similar experiment was performed in respect of ADAM17. The results are schematically shown in FIGS. 2 and 3, from which can be seen that nanobodies 35B1, 35C12, 32E1, 40A6 have an inactivating effect and nanobodies 35A12, 34H1 and 33G6 have an activating effect. The striking stabilizing effect that the presence of non-relevant protein has on the ADAM17 (see non-relevant 11F2 nanobody at 25× excess) should be noted.

A similar experiment was performed in respect of ADAM10. The results are schematically shown in FIG. 4, which shows the effect of purified nanobodies (400 nM, 10 molar excess) on the activity of recombinant ADAM10 (200 ng=40 nM, R&D system). As can be seen from FIG. 4, nanobodies 39D8, 39B1, 39D1 and 33D1 have an inactivating effect, and nanobodies 39F1, 32E1 (have related sequence=family 2) and 33G6, 39G8, 39H8, 39A8 and 39C8 (all part of the same family 1) have an activating effect. The experiment was repeated with some clones, with the results being schematically shown in FIGS. 5 and 6, which shows the effect of the increasing amount of purified nanobodies (excess relative to ADAM10) on the activity of recombinant ADAM10 (R&D system). As can be seen from FIG. 5, nanobodies 39D8, 39B1, 39D1 and 33D1 have an inactivating effect, and nanobodies 39C8, 32E1 and 39F1 have an activating effect. The controls (11F2 and 30E6) showed little to no effect (see also FIG. 6, which shows the effect of 50 molar excess of purified nanobodies on the activity of recombinant ADAM10 (R&D system).

The activity of amino acid sequences or polypeptides of the invention on ADAM-TS5 can be tested using an commercially available ELISA-based kit (from Invitek). For testing the activity of amino acid sequences or polypeptides of the invention on ADAM10 in a cell-based assay, cleavage of CD44 into the medium of cell can be used to detect the activity of ADAM10 in U251 glioblastoma (S. Atkinson and G. Murphy, publication submitted). For testing the activity of amino acid sequences or polypeptides of the invention on ADAM17 in a cell-based assay, the release of HB-EGF conjugated Alkaline phosphatase from transfected MCF7 cells can be used a readout for ADAM17 activity (S. Atkinson and G. Murphy, submitted).

For testing the activity of amino acid sequences or polypeptides of the invention on ADAM10 in a cell-based assay, cleavage of CD44 into the medium of cell can be used to detect the activity of ADAM10 in U251 glioblastoma (S. Atkinson and G. Murphy, publication submitted). For testing the activity of amino acid sequences or polypeptides of the invention on ADAM17 in a cell-based assay, the release of HB-EGF conjugated Alkaline phosphatase from transfected MCF7 cells can be used a readout for ADAM17 activity (S. Atkinson and G. Murphy, submitted).

Table B-2 gives the amino acid sequences of a number of Nanobodies against some representative ADAM proteinases.

TABLE B-2 Nanobodies against some representative ADAM metalloproteinases. SEQ ID NO's 868 to 886: Nanobodies against ADAM8 SEQ ID NO's 887 to 907 and 1044 to 1047: Nanobodies against ADAM9 SEQ ID NO's 908 to 931: Nanobodies against ADAM10 SEQ ID NO's 932 to 948 and 1048 to 1052: Nanobodies against ADAM17 SEQ ID NO's 949 to 974 and 1053: Nanobodies against ADAMTS5 >28-B12, C12, D6, E12 SEQ ID NO: 868 EVQLVESGGGLVQNGGSLRLSCVMSGSSISLYTSGWYRQAPGK QREWVASIASGASGGTTVYEDSVKGRFTISRDDAENTVYLQMITLKPE DTAVYYCRAQDPMRTRDAYWGQGTQVTVSS >28-D1, H1 SEQ ID NO: 869 EVQLVESGGGLVQAGGSLRLSCAASGRTFDNYIMGWFRQAPG KEREFVAAINYEGDRTYSPNSVKGRFTISRDNAKNTVYLQMNSLKPED TAVYQCATGPRYGKYDYWGQGTQVTVSS >28-A6 SEQ ID NO: 870 EVQLVESGGGLVQAGGSLRLSCAASGRTFSDYTMYWFRQAPG KERELVAAIGGVTDYADSVKGRFTIARDNAKSTVSLQMNSLKPEDTAV YYCAAKHRSVATRTGGNVSWGQGTQVTVSS >28-B6 SEQ ID NO: 871 EVQLVESGGGLVQAGASLKISCAASGRTYDIRVMGWFRRAPG KDRESVATVTWRDNITYYVDSVKGRFTITRDNAKNTVYLQMNNLKPE DTAVYYCAAQTEDSAQYIYWGQGTQVTVSS >28-C1 SEQ ID NO: 872 EVQLVESGGGLVQAGGSLRLSCAASGRTFSNYVMGWFRQAPG REREFVAAMGDTTLYADSVEGRFTISRDNDQNTVYLQMSSLKPEDTA AYFCAARSRFIPPLTFRTGGAYDYWGQGTQVTVSS >28-C6 SEQ ID NO: 873 EVQLVESGGGLVQAGASLKLSCAASGRTYDIRVMGWFRRAPG KGRESVATTTWRDNITYYMDSVKGRFTITRDNAKNTVYLQMNNLKPE DTAVYYCAAQTEDSAQYIYWGQGTQVTVSS >28-D12 SEQ ID NO: 874 EVQLVESGGGLVQAGGSLRLSCATSGSIASFNAMAWHRQAPGN QRELVAAIHISGNTNYADSVKGRFTISRDNGKNTVYLQMNSLRPEDTA VYYCNAIEVRTGLSPRGHWGQGTQVTVSS >28-E1 SEQ ID NO: 875 EVQLVESGGGLVQAGGSLRLSCTASGRTFSDYTMAWFRQAPG KERELVAAIGSVTDYADSVKGRFTISRDNAKNTVSLQMNSLKPEDTAV YYCAAKHRTRTRGYVNWGQGTQVTVSS >28-E6 SEQ ID NO: 876 EVQLVESGGGLVQAGGSLRLSCAASGNIFRIETMAWHRQAPGK QRELVAAIRSDDMTNYPDSVKGRFTISRDNFKNTVYLQMNSLTPEDTA VYYCNLIQRRAPYSRLETYWGQGTQVTVSS >28-F12 SEQ ID NO: 877 EVQLVESGGGLVQAGESLRLSCKASRNDFSFNSMAWYRQSPGK QRNLVARIFRSANIYYDDSVKGRFTISRDSALNTVFLQMNALKSEDTA VYYCNGRLSNGLDYWGQGTQVTVSS >28-G1 SEQ ID NO: 878 EVQLVESGGGLVQAGGSLRLSCAASGNIFRIETMAWHRQAPGK QRELVAAIRRDEMTNYLDFVKGRFTISRDNFKNTVYLQMNSLKPEDTA VYYCNLIQRRAPYSRLETYWGQGTQVTVSS >28-G12 SEQ ID NO: 879 EVQLVESGGGLVQAGGSLRLSCAASGSIFSINTMGWYRQTPGQ QRDWVASISSGGTTAYADSVKGRFSISRDGPKNTVYLQMNSLKPEDTA VYYCKAQRRWSQDYWGQGTQVTVSS >28-G6 SEQ ID NO: 880 EVQLVESGGGLAQAGGSLRLSCAASGSITSFNAVGWWRQAPGT QREWVASISTSGSTITPYVDSVKGRFTISRDNTKNTVYLQMNSLKPEDT AVYYCCATERMHHSYCGQGTQVTVSS >29-A12 SEQ ID NO: 881 EVQLVESGGGLVQAGGSLRLSCAASGRTFSEVVMGWFRQTPG MKREFVAAISGSENVTSYADSVKGRFTISRDNAKNTVTLQMNSLKPED TAVYYCAAQRWRGGSYEYWGQGTQVTVSS >29-A6 SEQ ID NO: 882 EVQLVESGGGLVQAGGSLGLSCVFSGRPYSYDAMAWFRQAPG KEREFVAVITWSGGTTVYADSVQGRFTISRDIAKNTMNLQMNSLKPDD TAVYYCAGSAGARYGVGWWRNGQNYQNWGQGTQVTVSS >29-C6 SEQ ID NO: 883 EVQLVESGGESVQVGGSVRLSCAASGLTFSNFIMGWFRRAPGK ERELLAAIGDPSTHYADSATGRFTISRDNAKNMVYLQMNSLKTEDTAV YYCAARSRYSTGTLYDQTKYIYWGQGTQVTVSS >29-E12 SEQ ID NO: 884 EVQLVESGGGLVQAGGSLTLSCVVSGVDFSRHVIGWFRQAPGK ERKFVTAINSDGDTTTDDRSLKGRFTISRFNANNTVHLQMTNLKVEDT AIYYCATGPQYRSYFARSYLYWGQGTQVTVSS >29-F6 SEQ ID NO: 885 EVQLVESGGGRVQAGGSLRLSCEASGRTFSDYIIGWFRQGPGKE RESVARISGSGLTNTTTETVKGRFTISRDNAKNTVYLQMNSLKPEDTA VYYCAAAYSGHFSGRVSDFLYWGQGTQVTVSS >29-H1 SEQ ID NO: 886 EVQLVESGGGLVQPGGSLRLACAASKSIDNIHAMGWYRQAPGN EREWVASITSSATAYADSVKGRFIISRDNAENTIYLQMHSLKPEDTAVY YCKGQVLVPGGRNDYWGQGTQVTVSS >41-B5, C1, D3, E4, E5 SEQ ID NO: 887 EVQLVESGGGLVQAGGSLRLACAASGRTFSTYAMGWFRQAPG KEREFVAAISWNEVNTYYTDSVKGRFTISRNNAENTVYLQMNSLKRE DTAVYYCASDRHYTAQQMRVMTGASYMDYWGKGTLVTVSS >30-B 12, E6 SEQ ID NO: 888 EVQLVESGGGLVQAGGSLRLSCAASGPTFSIYDMGWFRQAPGK EREFVTVIRWSDGFTYYEDSVKGRFTISRDNAKNTVYLQMNNLKPEDT AVYVCAANTAPLRIINFRNAYNYDYWGQGTQVTVSS >30-A6 SEQ ID NO: 889 EVQLVESGGGLVQAGGSLRLSCAASGRASGDYAMGWFRQAPE KEREFVATINYSGGVTYYADSVKGRFTISRDNAKNTVYLQMNSLKPED TAVYYCAADRHYGAYALLGLGTHAYIDYRGQGTQVTVSS >30-B1 SEQ ID NO: 890 EVQLVESGGGLVPAGGSLRLSCAGSGRSFSRLAMGWFRQAPGK ERDFVGAINWLSESTYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCASDRFATAQNMNTMGSLDYWGQGTQVTVSS >30-C1 SEQ ID NO: 891 EVQLVESGGGLVQAGDSLRLSCAVSGRTISSYAVGWFRQAPGK EREFVAVISWTGGSTYFADSVKGRFTISRDNAKNTVYLQMNNLKSEDT AVYYCTADRFTTGSGRTSYPRPSEFDHWGQGIQVTVSS >30-D1 SEQ ID NO: 892 EVQLVESGGGLVQPGGSLRLSCAASGFAFSSDWMYWVRQAPG KGPEWVSSIGIAGTPTFYADSVKGRFTISRDNANNMLYLQMTSLKPGD TALYYCAREGIYCSNWRCLFGPKTDLPASWGQGTQVTVSS >30-D12 SEQ ID NO: 893 EVQLVESGGGLVQAGGSLRLSCATSGRTFSDYVVGWFRQTPGK EREFIGRKVWSNGNTYYIDSVKGRFTISGDNAKRTVYLQMNSLKPEDT ALYYCAARSPMSPTWDNWGQGTQVTVSS >30-E12 SEQ ID NO: 894 EVQLVESGGGLVPAGGSLRLSCAGSGRTFSRLAMGWFRQAPGK EREFVAAISWLGESTYYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCASDRFATAQAMGAMGSLDYWGQGTQVTVSS >30-G1 SEQ ID NO: 895 EVQLVESGGGLVQAGISLKLSCTASGGTLTNYFMGWFRQAPGK EREFVAGVAWSSDFTAYTDSVKGRFTISRDNAKNTVYLQMNDVKPED TAVWYCAARRRGGGYNQLNLYDYWGQGTQVTVSS >30-G12 SEQ ID NO: 896 EVQLVESGGRLVQAGGSLRLSCAASGRTFSTDVMGWFRQVPEK EREFVAEIGWRDTTLYADSVKGRFTISRDNAKNMVYLQMNSLKPEDT AVYYCSSRRAAAAYDQWGQGTQVTVSS >31-B1 SEQ ID NO: 897 EVQLVESGGGLVQAGGSLRLACAASGRTFSTLAMGWFRQAPG KEREFVAAISWSEVNTYYTDSVKGRFTISRNNAENTVYLQMNSLKPED TAVYYCAADRHYSAQQMRVMTGASYMDYWGKGTLVTVSS >31-D12 SEQ ID NO: 898 EVQLVESGGGLVQAGDSLRLSCAPSGGIISNYHVGWFRQAPGK EREFVAAISASGTSTAYAGSVKGRFTISRDNAKNTAFLQMNTLKPEDT AVYYCAASEYVFRYYDDSRYYAYWGQGTQVTVSS >31-E12 SEQ ID NO: 899 EVQLVESGGGLVQAGGSLRLSCSSSGRTYSAYNMGWFRLRPGK EREFVAAINWSGGTQDYVDSVKGRFTAIADVAKKTVFLQMTSLKPED TAVYYCAATQWGSSGWKQARWYDFWGQGTQVTVSS >31-F6 SEQ ID NO: 900 EVQLVESGGGLVQAGGSLRLACAASGRTFSTLAMGWFRQAPG KEREFVAAISWSEVNTYYTDSVKGRFTISRNNAENTVYLQMNSLKRED TAVYYCASDRHYTAQQMRVMTGASYMDYWGKGTLVTVSS >31-G12 SEQ ID NO: 901 EVQLVESGGGLVQAGESLRLSCAASGRTFGNYVMGWFRQAPG KERELVAAITWSDSRTDYADSVKGRFTISRDNVKNLVYLQMNSLRPED TAVYSCAASSIGPYRLLDSSRYAYWGRGTQVTVSS >41-A1 SEQ ID NO: 902 KVQLVESGGGLVQAGGSLKLACAASGRTFSTLAMGWFRQAPG KEREFVAAISWSEVNTYYTDSVKGRFTISRNNAENTVYLQMNSLKPED TAVYYCAADRHYSAQQMRVMTGASYMDYWGKGTLVTVSS >41-A3 SEQ ID NO: 903 EVQLVESGGGLVQAGGSLRLACAASGRTFSTYAMGWFRQAPG KEREFVAAISRNEVNTYYTDSVKGRFTISRNNAENTVYLQMNSLKRED TAVYYCASDRHYTAQQMRVMTGASYMDYWGKGTLVTVSS >41-B1 SEQ ID NO: 904 EVQLVESGGGLVQAGGSLRLSCTGSDRTFIGYHMGWFRQAPGK EREFVAYISSGGAYSNYADSVKGRFTISRDNAKNTAYLQMNSLKPEDT AVYHCAATDYNKAYAREGRRYDYWGQGTQVTVSS >41-C5 SEQ ID NO: 905 EVQLVESGGGLVQAGGSLRLSCVVSGTITSAVFMGWYRQLPGK QRELVASINRGGSTNYAPSAKGRFTISRVNANSTMYLQMNSLQPEDTA VYYCYGVINTRSFWGQGTQVTVSS >41-D1 SEQ ID NO: 906 EVQLVESGGGLVQAGGSLRLACAASGRTFSTLAMGWFRRAPG KEREFVAAISWSEDNTYYSDSVKGRFTISRNNAESTVYLQMNSLKPED TAVYYCASDRHLLAQQMRVMTGASYMDYWGKGTLVTVSS >41-F6 SEQ ID NO: 907 EVQLVESGGGLVQAGGSLRLACAASGRTFSTLAMGWFRQAPG KEREFVAAISWSEVNTYYTDSVKGRFTISRNNAENTVYLQMNSLKPED TAVYYCVSDRHATAQHMRVMTGASYMDYWGKGTLVTVSS >39-A8, D7, E7, F9, G8, H8, A9 SEQ ID NO: 908 EVQLVESGGGLVQTGGSLRLSCAASGRTFTSYCVGWWRQAPG KERDVVAAITRGSNSTDYVDSVKGRFTISRDNAENTVYLQMNSLKPED TAVYYCAADTNCRNLYTGRPEYWGQGTQVTVSS >32-E12, G12, 39-D2, F2 SEQ ID NO: 909 EVQLVESGGGLVQAGGSLRLSCAASGFTFGDYAIGWFRQAPGK EREGVSCISISDSSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTA VYYCAADRLAYGLDPNFYDYWGQGTQVTVSS >32-E1, E6, 39-A2 SEQ ID NO: 910 EVQLVESGGGLVQAGGSLRLSCAASERIFSTYFMGWFRQAPGK EREFVAFISGNGGSTDYADSVKGRFAISRDNVKNTLYLQMSSLKPDDT AVYYCAVAGRQIKSTWDYWGQGTQVTVSS >32-D1, 39-A1 SEQ ID NO: 911 EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGK EREGVSCISVSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDT AVYYCAADRLAYGLDPNFYDHWGQGTQVTVSS >39-D8, E8 SEQ ID NO: 912 EVQLVESGGGLVRARGSLRLSCAASGGTFSRYAMGWFRQAPG KEREFVAAISRSGGNTYFTESVKGRFTMSRDNAKNTVYLQMNSLKPED TAVYYCAARSAAVYTTTVYLVDFEYNYWGQGTQVTVSS >32-C6 SEQ ID NO: 913 KVQLVESGGGLVQAGGSLRLSCAASGFTFGDYAIGWFRQAPGK EREGVSCISISDSSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDTA VYYCAADRLAYGLDPNFYDYWGQGTQVTVSS >32-F1 SEQ ID NO: 914 EVQLVESGGGLVQAGGSLTLSCAASGRTFSSYRLGWFRQAPGN EREFVASITWSSANTYYADSVKGRFTISRERAKNTMYLQMDSLRPEDT AVYYCAKEDVGKPFDSWGQGTQVTVSS >32-G6 SEQ ID NO: 915 EVQLVESGGGLVQPGGSLRLSCAASGFTFDDYAIGWFRQAPGK EREGVSCISSSDGRTYYDDSVKGRFTISSDNAKNTVYLQMNSLKPEDT AVYYCAADRLAYGLDPNFYDYWGQGTQVTVSS >33-A1 SEQ ID NO: 916 EVQLVESGGGLVQAGGSLRLSCAASGDTFSRYSMGWFRQAPG KEREFVVYITRSGRTTYYQDSVKGRFTISRDNAKNTVYLQMNSLKPED TAVYYCAAMRSGNVRYPERYDYWGQGTQVTVSS >33-C6 SEQ ID NO: 917 EVQLVESGGGLVQAGDSLRLSCAASGRTFSSYYMGWFRQAPG KEREFVAHISLSGGNTEYADSVKGRFTITRDNAGNTVYLQMNSLKPED TGVYCCAASPSLRSAWQYWGQGTQVTVSS >33-D1 SEQ ID NO: 918 EVQLVESGGGLVQAGDSLRLSCAATGRTFSSDAMGWFRQAPG KERAFVAGINYNSVYRYYTDSVEGRFTISRDNAKNTVYLQMNSLKPED TAVYYCAADSDYYSLIGGRPVNYWGQGTQVTVSS >33-G6 SEQ ID NO: 919 EVQLVESGGGSVQTGGSLRLSCAASGRTFTSYCVGWWRQAPG KERAVVAAITRGGDTTDYVDSVKGRFTISRDKAENTVYLQMNSLKPE DTAVYYCAADINCRNLYTGRPEYWGRGTQVTVSS >39-B1 SEQ ID NO: 920 EVQLVESGGGLVQAGGSLRLSCARSGRISNINIMAWYRQAPGK TRDMVAAIIGDSTNYADSVKGRFTISRDNAKNTVYLHMNRLKPEDTG VYYCKISGVDWGQGTQVTVSS >39-B2 SEQ ID NO: 921 EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGK EREGVSCISSSDGRTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDT AVYYCAADRLAYGLDPNFYDYWGQGTQVTVSS >39-C1 SEQ ID NO: 922 EVQLVESGGGLVQAGGSLRLSCSASGRTFGSYVMGWFRQAPG KEREFVAAVSRSGRNINYADLMKGRFTVSRDNTKNTVYLQLNNLTPE DTAVYYCAADGTISSSWADLRRGETYGDWGQGTQVTVSS >39-C2 SEQ ID NO: 923 KVQLVESGGGLVQAGGSLRLSCAASGRTFSRYYMGWFRQAPG KEREFVAFISGTGGSIDYADSVKGRFTISRDSAKNTVYLQMNSLKPEDT AVYYCAAASGNSGRSTWDYWGQGTQVTVSS >39-E1 SEQ ID NO: 924 EVQLVKSGGGLVQAGGSLRLSCAASGNIFINNAVGWYRQAPGK QREMVAAMLSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCNVQVNGIWARWGQGTQVTVSS >39-E2 SEQ ID NO: 925 EVQLVESGGGLVQAGGSLRLSCGRSGRISNTNIMSWYRQAPGKT RDMVAAIIGDATNYADSVKGRFTISRDNAKNAVSLHMNRLKPEDTGV YYCKIPGVDWGQGTQVTVSS >39-F1 SEQ ID NO: 926 EVQLVESGGGLVQAGGSLRLSCAASERIFSTYFMGWFRQAPGK EREFVAFISGNGGSTDYADSVKGRFAISRDNVKNTLYLQMSSLKPDDT AVYYCAVAGRQIKSTWGYWGQGTQVTVSS >39-G1 SEQ ID NO: 927 EVQLVESGGGLVQAGGSLRLSCARSGRISNINIMSWYRQAPGKT RDMVAAIIGDSTNYADSVKGRFTISRDNAKNTVHLQMNRLKPEDTGV YYCNIPGVDWGQGTQVTVSS >39-G2 SEQ ID NO: 928 EVQLVESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGK EREGVSCISMSDGSTYYADSVKGRFTISSDNAKNTVYLQMNSLKPEDT AVYYCAADRLAFGLDSNFYDYWGQGTQVTVSS >39-C8 SEQ ID NO: 929 EMQLVESGGGLVQTGGSLRLSCAASGRTFTSYCVGWWRQAPG KERDVVAAITRGSNSTDYVDSVKGRFTISRDNAENTVYLQMNSLKPED TAVYYCAADINCRNLYTGRPEYWGQGTQVTVSS >39-D9 SEQ ID NO: 930 EVQLVESGGGLVQPGGSLRLACAASGRTFTSYCVGWWRQAPG KERDVVAAITRGSNSTDYVDSVKGRFTISRDNAENTVYLQMNSLKPED TAVYYCAADINCRNLYTGRPEYWGQGTQVTVSS >39-D1 SEQ ID NO: 931 KVQLVESGGGLVQAGGSLRLSCAASGNIFINNAVGWYRQAPGK QREMVAAMLSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCNVQVNGTWARWGQGTQVTVSS >35-B12, E1, G1, F6 SEQ ID NO: 932 EVQLVESGGGLVQTGGSLRLSCAASGGTFSTYAMGWFRQAPG KEREFVAGITRSAVSTSYTDSVKGRFTISRDNAKDMVYLQMNSLKPED TALYYCAATRRLHFGNGADYWGKGTPVTVSS >35-C6, 40-E1 SEQ ID NO: 933 KVQLVESGGGLVQTGGSLRLSCAASGGTFSTYAMGWFRQAPG KEREFVAGITRSAVSTSYTDSVKGRFTISRDNAKDMVYLQMNSLKPED TALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-A2, H2 SEQ ID NO: 934 EVQLVKSGGGLVQTGGSLRLSCIVSGGTFSTYAMGWFRQAPGK EREFVAGITRSGLSTSYADSVKGRFTISRDNAKDTVYLQMNSLKPEDT ALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-B1, D1 SEQ ID NO: 935 EVQLVESGGGLVQTGGSLRLSCIVSGGTFSTYAMGWFRQAPGK EREFVAGITRSGLSTSYADSVKGRFTISRDNAKDTVYLQMNSLKPEDT ALYYCAATRRLHFGNGADYWGKGTPVTVSS >35-B1, G12, 40-E2 SEQ ID NO: 936 EVQLVKSGGGLVQTGGSLRLSCAASGGTFSTYAMGWFRQAPG KEREFVAGITRSGVSTSYADSVKGRFTISRDNAKDMVYLQMNSLKPED TALYYCAATRRLHFGNGADYWGKGTPVTVSS >35-C12 SEQ ID NO: 937 KVQLVESGGGLVQTGGSLRLSCAASGGTFSTYAMGWFRQAPG KEREFVAGITRSAVSTSYTDSVKGRFTISRDNAKDMVYLQMNSLKPED TALYYCATTRRLHFGNGADYWGKGTPVTVSS >35-F12 SEQ ID NO: 938 EVQLVESGGGLVQTGGSLRLSCAASGGTFSTYAMGWFRQAPG KEREFVAGITRSGVSTSYADSVKGRFTISRDNAKDMVYLQMNSLKPED TALYSCAATRRLHFGNGADYWGKGTPVTVSS >35-G6 SEQ ID NO: 939 EVQLVESGGGLVQAGDSLRLSCTASGAFGSYAIAWFRQTPGKE REFVAVINRSGSYVYYTDSVKGRFTISRHNAKNTAYLQMNSLKPEDTA VYYCAARDGTLYSTTYYYISSYTYWGQGTQVTVSS >40-A1 SEQ ID NO: 940 EVQLVESGGGLVQAGGSLRLSCAASGGTFSTYAMGWFRQAPG KEREFVAGITRSAVSTSYTDSVKGRFTISRDNAKDMVYLQMNSLKPED TALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-B2 SEQ ID NO: 941 EVQLVESGGGLVQTGGSLRLSCIASGGTFSTYAMGWFRQAPGK EREFVAGITRSALSTSYADSVKGRFTISRDNAKDMVYLQMNSLKPEDT ALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-C1 SEQ ID NO: 942 EVQLVESGGGLVQAGDSLRLSCTASGTFGSYAIAWFRQTPGKE REFVAVINRSGSYVYYTDAVKGRFTISRHNAKNTAYLQMNSLKPEDTA VYYCAARDGTLYSTTYYYISSYTYWGQGTQVTVSS >40-D2 SEQ ID NO: 943 EVQLVESGGGLVQPGGSLRLSCRASGGTFRKLAVAWFRQAPGK EREFVAGITRSAVSTSYTDSVKGRFTISRDNAKDMVYLQMNSLKPEDT ALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-F1 SEQ ID NO: 944 EVQLVESRGGLVQTGGSLRLSCAASGGTFSTYAMAWFRQAPG KEREFVAGITRSAVSTSYADSVKGRFTISRDNAKDMVYLQMNSLKPED TALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-F2 SEQ ID NO: 945 EVQLVESGGGLVQTGGSLRLSCAASGGTFSTYAMGWFRQAPG KEREFVAGITRSSVSTSYADSVKGRFTISRDNAKDMVYLQMNSLKPED TALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-G1 SEQ ID NO: 946 EVQLVESGGGLVQAGDSLRLSCTASGTFGSYAIAWFRQTPGKE REFVAVINRSGSYMYYIDSVKGRFTISRHNAKNTAYLQMNSLKPEDTA VYYCAARDGTLYSTSYYYISSYTYWGQGTQVTVSS >40-G2 SEQ ID NO: 947 EVQQVESGGGLVQTGGSLRLSCIVSGGTFSTYAMGWFRQAPGK EREFVAGITRSGVSTSYADSVKGRFTISRDNAKDTVYLQMNSLKPEDT ALYYCAATRRLHFGNGADYWGKGTPVTVSS >40-H1 SEQ ID NO: 948 EVQLMESGGGLVQTGGSLRLSCAASGGTFSTYAMGWFRQAPG KEREFVAGITRSAVSTSYTDSVKGRFTISRDNAKDMVYLQMNSLKPED TALYYCAATRRLHFGNGADYWGKGTPVTVSS >36-A6, 40-H8 SEQ ID NO: 949 EVQLVESGGGLVQAGDSLRLSCAASGRTLSSYNMGWFRQAPG KEQEFVAADMWSGTTTYYTDSVKGRFTISRDNAKNMVYLQMNSLKP EDTAVYYCAAELHSSDYTSPGAYAYWGQGTQVTVSS >36-A1 SEQ ID NO: 950 EVQLVESGGGLVQPGGSLTLSCAASGGTFSIRAMGWLRQAPGN EREFVAAISRDGDRTYYTDVVKGRFTISRDNAKSTVYLQINSLKTEDTA VYYCAATRPFKVLTATIENDFTYWGQGTQVTVSS >36-C6 SEQ ID NO: 951 EVQLVESGGGLVQAGGSLRLSCAFSDGRTVSRYHMGWFRQGP GKEREFVAAVSSLGPFTRYADSVKGRFTISRDNAKNAVYLQMNSLKPE DTAVYYCAADSSGYSGSYSSEYRYDYWGQGTQVTVSS >36-D6 SEQ ID NO: 952 EVQLVESGGGLVQAGGSLRLSCVASGLTFRNYAMAWFRQAPG KEREFVAGINWSGNGVYYPDSLKERFAISRENPKNMVYLQMNSLNPE DTAVYYCTADRTLTAWDRDNAEYWGQGTQVTVSS >36-E1 SEQ ID NO: 953 EVQLVESGGGLVQAGDSLRLSCAASGRTLSSYNMGWFRQAPG KELEFVAAIMWSGTTTYYTDSVKGRFTISTDNAKNMVYLQMNSLKPE DTAVYYCAAELHSSDYTSPGAYAYWGQGTQVTVSS >36-F1 SEQ ID NO: 954 EVQLVESGGGLVQPGGSLTLSCAASGGTFSIRAMGWFRQAPGN EREFVAAISRDGDRTYYTDVVKGRFTISRDNAKSTVYLQINSLKTEDTA VYYCAATRPFKVLSAIIENDFTYWGQGTQVTVSS >37-B1 SEQ ID NO: 955 EVQLVESGGGLVQPGQSLRLSCAASGRTLNPYKVAWFRQAPGK ERDFVAVIHWYGITAYADTVKGRFSISRDNAKGTVYLQMDSLKPEDT AVYYCALDSTSALGHATTDFDSWGQGTQVTVSS >37-B12 SEQ ID NO: 956 EVQLVESGGGLVQAGGSLRLSCAASGRSLRNYHMAWFRQAPG KEQEFVGDFRPSGGSPYYANYADSVKGRFTIFRDNAKNTVYLQMNSL KLEDTAVYYCAADSHGGIAFMEPDEYDYWGQGTQVTVSS >37-B6 SEQ ID NO: 957 EVQLVESGGGLVQAGGSLRLSCTAAGRTHSIYPIGWFRQAPGKE REFVAAIDWSGSRSYYLDSMKGRFTISGDNAKNTVYLQMNSLKPEDT GVYYCAARPVGDFADPRYRFWGQGTQVTVSS >37-C12 SEQ ID NO: 958 EVQLVESRGGLVQTGGSLRLSCTTSGGTLRGYGVGWFRQGPGK DREFVAAISWSPGRTDYGDAVKGRFTISRDNAKNTVYLQMNSLKPED TAVYYCAADSVSASYYDARNDMAYDYWGRGTQVTVSS >37-C6 SEQ ID NO: 959 EVQLVESGGGVVQTGGSLRLSCTTSGGTFRNYGVGWFRQGPG RDREFVAAISWSPGRTDYGDAVKGRFTISRDNPKNTVYLQMNSLKPED TAVYYCAADSVSASYYDARNDMAYDYWGRGTQVTVSS >37-D6 SEQ ID NO: 960 EVQLVESGGGLVRAGGSLRLSCAASGRTLNPYKVAWFRQAPG KERDFVAVIHWYGITAYADTVKGRFSISRDNAQGTVKLQMDSLKPED TAVYYCALDSTSALGHTTSDFDSWGQGTQVTVSS >37-E6 SEQ ID NO: 961 EVQLVESGGGLVQAGGSLRLSCAASGRSLGTYHMAWFRQAPG KEQEFVGDLRPSGGRAGYADYADSVKGRFTIFRDNAKNTVYLQMNSL KLEDTAVYYCAADSHGGISFMEPDEYDYWGQGTQVTVSS >37-F1 SEQ ID NO: 962 KVQLVESGGGLVRAGGSLRLSCAASGRTLNPYKVAWFRQAPG KERDFVAVIHWYGITAYADTVKGRFSISRDNAKGTVYLQMDSLKPED TAVYYCALDSTSALGHATTDFDSWGQGTQVTVSS >37-F12 SEQ ID NO: 963 EVQLVESGGGLVQTGGSLRLSCTTSGGTFRNYGVGWFRQGPGK DREFVAAISWSPGRTDYGDAVKGRFTISRDNAKNTVYLQMNSLKPED TAVYYCAADSVSASYYDARNDMAYDYWGRGTQVTVSS >37-G1 SEQ ID NO: 964 EVQLVESGGGLVQAGGSLRLTCAASGRTFSMGRVGWFRQDPG KEREFVAAISRSGDTTYYDDSVRDRFTTSRDNAKNTIDLRMNSLKPED TAVYYCAAATFRAVRQDPSYSASYDYWGQGTQVTVSS >37-G6 SEQ ID NO: 965 EVQLVESGGGLVRAGGSLRLSCAASGRTLNPYKVAWFRQAPG KERDFVAVIHWYGITAYADTVKGRFSISRDNAKGTVYLHMDSLKPED TAVYYCALDSTSALGHATTDFDSWGQGTQVTVSS >40-A7 SEQ ID NO: 966 EVQLVESGGGLVQAGASLRLSCGASGRTLSMYTMGWFRQAPG KERDFVAAITPINWGGRGTHIADSVKGRFTIFRDNTKNTINLQMNNLNP EDTAVYYCAAESHGSTSPRNPLQYDYWGQGTQVTVSS >40-B8 SEQ ID NO: 967 EVQLVESGGGLVQAGGSLRLSCAASDSARTFTNYAIAWFRQAP GKERKFVAVVNWSTYYADSVKGRFTISRDNAKNTVNLQMNSLKPEDT AVYYCAVGDYRSDYRSPVAYNYWGQGTQVTVSS >40-D7 SEQ ID NO: 968 EVQLMESGGGLVQAGGSLRLSCAASGRTISSYTMAWFRQAPGK EREFVAYVFGGGEITDYADFVKGRFTISRDNAKNTGYLQMNSLKPEDT AVYYCAMECVGDVYRSRDYTYWGQGTQVTVSS >40-F7 SEQ ID NO: 969 EVQLVESGGGLVQAGGSLRLSCVASGLTFRNYAMAWFRQAPG KEREFVAGINWSGNGVYYPDSLKERFAISRENAKNMVYLQMNSLNPE DTAVYYCTADRTLTAWDRDSAEYWGQGTQVTVSS >40-F8 SEQ ID NO: 970 EVQLVESGGGLVQAGGSLRLSCAASGLTFRMYGSGWFRRAPG KEREFVGFINHSGGRTNYADSVKGRFTISRDNAKDTVYLQMNSLKPED TAVYYCAVPISGYINPAVYDRPGSYDYWGQGTQVTVSS >40-G7 SEQ ID NO: 971 EVQLVESGGGLVQAGGSLRLSCVASGLTFRNYAMAWFRQAPG KEREFVAGINWSGNGVYYPDSLKERFAISRENSKNMVYLQMNSLNPE DTAVYYCTADRTLTAWDRESAEYWGQGTQVTVSS >40-G8 SEQ ID NO: 972 EVQLVESGGGLVQAGASLRLSCGASGRTLNMYTMGWFRQAPG KERDFVAAITPINWGGRGTHIADSVKGRFTIFRDNTKNTIILQMNNLNP EDTAVYYCAAESHGSTSPRNPLQYDYWGQGTQVTVSS >40-H7 SEQ ID NO: 973 EVQLVESGGGLVQAGGSLGLSCAASGRTISSYTMAWFRQAPGK EREFVAYVFGGGEITDYADFVKGRFTISRDNAKNTGYLQMNSLKPEDT AVYYCAMECVGDVYRSRDYTYWGQGTQVTVSS 30-B6 SEQ ID NO: 1044 EVHLVESGGGVVQAGGSLRLSCVASGLPFSEYALVWFRQAPGK EKEREFVAGFAANGINTDYTDSVKGRFTISRDNAKNTVYLQMNSLKPE DTAVYYCAASWTPAYNDIPRHMSSMDQWGKGTLVTVSS 30-C6 SEQ ID NO: 1045 EVQLVESGGGLVQAGGSLRLSCAASGSIFSSNTMAWYRQAPRK QREFVASIMTDGTITYADSVKGRFAISRDTAKNTVALQMNSLKPEDTA VYYCNARQYGEYWQAAGSWGQGTQVTVSS x30-D6 SEQ ID NO: 1046 EVqLVESGGGLVQPGGSLRLSCAGSGRTFSRLAMGWFRQAPGK EREFVAAISWLAETTYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDT AVYYCASDRFATAQHMGAMGSLDYWGQGTQVTVSS 30-F12 SEQ ID NO: 1047 EVQLVESGGGLVQPGGSLRLSCAASGFTLDSYAIAWFRQAPGK EREGVSCISSSDGTTYYADSVKGRFTISRDNAKNMVYLQMQSLKPEDT AVYYCAADPELVTVCRPGWGPAYDYWGQGTQVTVSS 34-A12 SEQ ID NO: 1048 EVQLVESGGGLVQPGGSLRLSCAASGFPLDYYAIGWFRQAPGK EREGVSCISSSDGIRYYIDSVKGRFTISRDNAKNMVYLQMNSLKPEDTA VYYCAADPIRICSSQPRRYDYWGQGTQVTVSS 34-H1 SEQ ID NO: 1049 EVQLVESGGGLVQDGGSLSCAASGRTFSSYVMGWFRQAPGKE REFVATISRSGESTYYTGSVKGRFTISRDNAENTVYLQMNSLKPEDTAV YYCAADYNPYQSGSYYSSRSSTYDYWGQGTQVTVSS 35-A12 SEQ ID NO: 1050 EVQLVESGGGLVQAGGSLRLSCVASGLSFDDYAIGWFRQAPGK EREGVSCIGDKGDRIFYADSVKGRFAISSDHAKHTVDLQMTNLKPEDT ATYYCAAVAPFAFCTESLPDTWYDYWGQGIQVTVSS 35-D1 SEQ ID NO: 1051 EVQLVESGGGLVQAGGSLRLSCAASGRTFSTYAMAWFRRAPG KEREFVVAIRWSGDRTYYADSVKGRFTISRDNAKNTVYLQMNSLKPE DTAVYYCALRRFSGFDYSGNYYAWDAYDYWGQGTQVTVSS 35-H1 SEQ ID NO: 1052 EVQLVESGGGWVQAGDSLRLSCAASGRTFSSYAMGWFRQAPG KEREFVAGINGSGNGIRIALSVRGRFTITRDNAKNTGYLQMNSLKREDT AVYYCGPTFAAGASEYHYWGQGTQVTVSS x40-E8 SEQ ID NO: 1053 EVQLVESGGTLVXAGGSLRLSCAASGLAFNAYGTGWFRQAPG KEREFVATISWSGSTTSYADSVKGRFTVSRDNAKNTVYLQMNSLKPED TAIYYCAMNRYGLVYKEERHYDFWGQGTQVTVSS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in their entirety for the purpose and information indicated in the specification. 

We claim:
 1. A compound or VHH construct, that comprises or essentially consists of: a) an amino acid sequence that specifically binds to A Disintegrin And Metalloprotease Thrombospondin 5 (ADAMTS5), and inhibit the proteinase activity of ADAMTS5, and that essentially consists of a VHH sequence, which consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), in which: CDR1 is SEQ ID NO: 313, CDR2 is SEQ ID NO: 525 and CDR3 is SEQ ID NO: 737; CDR1 is SEQ ID NO: 314, CDR2 is SEQ ID NO: 526 and CDR3 is SEQ ID NO: 738; CDR1 is SEQ ID NO: 315, CDR2 is SEQ ID NO: 527 and CDR3 is SEQ ID NO: 739; CDR1 is SEQ ID NO: 316, CDR2 is SEQ ID NO: 528 and CDR3 is SEQ ID NO: 740; CDR1 is SEQ ID NO: 317, CDR2 is SEQ ID NO: 529 and CDR3 is SEQ ID NO: 741; CDR1 is SEQ ID NO: 318, CDR2 is SEQ ID NO: 530 and CDR3 is SEQ ID NO: 742; CDR1 is SEQ ID NO: 319, CDR2 is SEQ ID NO: 531 and CDR3 is SEQ ID NO: 743; CDR1 is SEQ ID NO: 320, CDR2 is SEQ ID NO: 532 and CDR3 is SEQ ID NO: 744; CDR1 is SEQ ID NO: 321, CDR2 is SEQ ID NO: 533 and CDR3 is SEQ ID NO: 745; CDR1 is SEQ ID NO: 322, CDR2 is SEQ ID NO: 534 and CDR3 is SEQ ID NO: 746; CDR1 is SEQ ID NO: 323, CDR2 is SEQ ID NO: 535 and CDR3 is SEQ ID NO: 747; CDR1 is SEQ ID NO: 324, CDR2 is SEQ ID NO: 536 and CDR3 is SEQ ID NO: 748; CDR1 is SEQ ID NO: 325, CDR2 is SEQ ID NO: 537 and CDR3 is SEQ ID NO: 749; CDR1 is SEQ ID NO: 326, CDR2 is SEQ ID NO: 538 and CDR3 is SEQ ID NO: 750; CDR1 is SEQ ID NO: 327, CDR2 is SEQ ID NO: 539 and CDR3 is SEQ ID NO: 751; CDR1 is SEQ ID NO: 328, CDR2 is SEQ ID NO: 540 and CDR3 is SEQ ID NO: 752; CDR1 is SEQ ID NO: 329, CDR2 is SEQ ID NO: 541 and CDR3 is SEQ ID NO: 753; CDR1 is SEQ ID NO: 330, CDR2 is SEQ ID NO: 542 and CDR3 is SEQ ID NO: 754; CDR1 is SEQ ID NO: 331, CDR2 is SEQ ID NO: 543 and CDR3 is SEQ ID NO: 755; CDR1 is SEQ ID NO: 332, CDR2 is SEQ ID NO: 544 and CDR3 is SEQ ID NO: 756; CDR1 is SEQ ID NO: 333, CDR2 is SEQ ID NO: 545 and CDR3 is SEQ ID NO: 757; CDR1 is SEQ ID NO: 334, CDR2 is SEQ ID NO: 546 and CDR3 is SEQ ID NO: 758; CDR1 is SEQ ID NO: 335, CDR2 is SEQ ID NO: 547 and CDR3 is SEQ ID NO: 759; CDR1 is SEQ ID NO: 336, CDR2 is SEQ ID NO: 548 and CDR3 is SEQ ID NO: 760; CDR1 is SEQ ID NO: 337, CDR2 is SEQ ID NO: 549 and CDR3 is SEQ ID NO: 761; or CDR1 is SEQ ID NO: 993, CDR2 is SEQ ID NO: 1013 and CDR3 is SEQ ID NO: 1033; and further comprises b) a serum protein, a VHH that specifically binds to human serum albumin, or an Fc portion.
 2. The compound or VHH construct according to claim 1, which is a multispecific construct.
 3. The compound or VHH construct according to claim 1, in which said serum protein is human serum albumin.
 4. The compound or VHH construct according to claim 1, that has a serum half-life that is at least 1.5 times greater than the half-life of the VHH sequence that specifically binds to ADAMTS5.
 5. The compound or VHH construct according to claim 1, that has a serum half-life that is increased than 1 hour compared to the VHH sequence that specifically binds to ADAMTS5.
 6. The compound or VHH construct according to claim 1, that has a serum half-life in human of at least about 12 hours.
 7. A composition, comprising at least one compound or VHH construct according to claim
 1. 8. The compound or VHH construct according to claim 1, wherein said VHH sequence is selected from the group consisting of SEQ ID NOs: 949-973 and
 1053. 9. The compound or VHH construct according to claim 1, wherein the serum protein, the VHH that specifically binds to human serum albumin, or the Fc portion is linked via a linker to the amino acid sequence that specifically binds to ADAMTS5.
 10. The compound or VHH construct according to claim 1, wherein the VHH sequence is a humanized VHH sequence, and wherein the framework region amino acid residue at position: 11 is selected from the group consisting of L, M, S, V, W; 37 is selected from the group consisting of F, Y, H, I, L and V; 44 is selected from the group consisting of G, E, A, D, Q, R, S, and L; 45 is selected from the group consisting of L, R, C, I, L, P, Q, and V; 47 is selected from the group consisting of W, L, F, A, G, I, M, R, S, V and Y; 83 is selected from the group consisting of R, K, N, E, G, I, M, Q and T; 84 is selected from the group consisting of P, A, L, R, S, T, D, and V; 103 is selected from the group consisting of W, P, R and S; 104 is selected from the group consisting of G and D; and 108 is selected from the group consisting of Q, L and R, with the proviso that when positions 44-47 are GLEW (SEQ ID NO:1054), position 108 is always Q in VHH sequences that also contain a W at 103; wherein the amino acid residue position is according to the Kabat numbering. 